**Part 3**

**Understanding Immunological Aspects of HIV in an Infected Person**

148 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

Yurin O., Kravchenko A., Afonina L., et al. (2001). Nikavir – a novel domestic antiretroviral

Zakharova N., Kuptsov D., Mishustina Y., et al. (2008). Experience of nikavir (phosphazide)

application in chemoprevetion of the vertical HIV transmission. *Infectious Diseases*,

agent. Epidemiology and Infectious diseases, № 1, p. 43-45.

Anthology, St.Petersburg, 2007, p. 93-98.

**9**

*Russia*

**Natural Catalytic Antibodies**

Georgy A. Nevinsky

 **in Norm and in HIV-Infected Patients**

*Institute of Chemical Biology and Fundamental Medicine of SD of RAS*

Antibodies (Abs) have been first characterized as proteins which are produced by the immune system and have a sole function of binding other molecules, called antigens, with the goal of eliciting an immune response. In this classical conception, Abs act similarly to enzymes in specific binding other molecules. However, in contrast to enzymes they do not have the ability to catalyze chemical conversions of their bound partners. For the vast majority of Abs, this observation is correct. However, in a 1946 consideration of enzyme function, Linus Pauling first hypothesized that the active center of an enzyme is closely juxtaposed to a "strained configuration" of its substrate (that is, targeted against the structure of the transition state) rather than to the native conformation of the substrate molecule (Pauling, 1946). This idea led Jencks in 1969 to propose that Abs generated in an anti-hapten immune response against chemically stable analogs of the transition-state of a

reaction of interest could potentially display an enzymatic activity (Jencks, 1969).

anti-hapten catalytic Abs were termed abzymes (derived from **a**nti**b**ody en**zyme**).

activity (Barbas et al., 1997; Janda et al., 1997; Keinan, 2005, and refs therein).

In 1985, a general method for generating catalytic monoclonal Abzs against transition state analogs, and a way to use those Abs to accelerate chemical reactions, was first described (Schochetman & Massey, 1985). One year later two groups were able to produce the first monoclonal Abs with catalytic properties, which were generated against hapten analogs of the transition states for *p*-nitrophenylphosphorylcholine (Pollack et al., 1986) or for monoaryl phosphonate esters (Tramontano et al., 1986a, 1986b). The artificial monoclonal

The evolution of the technology of artificial Abzs during the last two decades has led not only to the rapid development of direct approaches for the generation of Abs with specified properties, but also to the creation of strategies to revise the targeting specificity of individual Abzs. Such modifications of antigen binding specificity can be achieved genetically *in vitro*, by application of the site-directed mutagenesis, or genetic selection or screening (using approaches such as phage display). Alternatively, modification can be induced directly on purified antibody, *via* selective chemical modification by direct introduction of catalytic groups into the Ab combining site. Some studies describing these approaches include (Keinan, 2005, and refs therein). The employment of the approaches have demonstrated that the substrate specificity (and/or the specific activity) of some artificial Abzs is comparable to or even higher than that of enzymes with the same catalytic

**1. Introduction**

## **Natural Catalytic Antibodies in Norm and in HIV-Infected Patients**

Georgy A. Nevinsky *Institute of Chemical Biology and Fundamental Medicine of SD of RAS Russia*

## **1. Introduction**

Antibodies (Abs) have been first characterized as proteins which are produced by the immune system and have a sole function of binding other molecules, called antigens, with the goal of eliciting an immune response. In this classical conception, Abs act similarly to enzymes in specific binding other molecules. However, in contrast to enzymes they do not have the ability to catalyze chemical conversions of their bound partners. For the vast majority of Abs, this observation is correct. However, in a 1946 consideration of enzyme function, Linus Pauling first hypothesized that the active center of an enzyme is closely juxtaposed to a "strained configuration" of its substrate (that is, targeted against the structure of the transition state) rather than to the native conformation of the substrate molecule (Pauling, 1946). This idea led Jencks in 1969 to propose that Abs generated in an anti-hapten immune response against chemically stable analogs of the transition-state of a reaction of interest could potentially display an enzymatic activity (Jencks, 1969).

In 1985, a general method for generating catalytic monoclonal Abzs against transition state analogs, and a way to use those Abs to accelerate chemical reactions, was first described (Schochetman & Massey, 1985). One year later two groups were able to produce the first monoclonal Abs with catalytic properties, which were generated against hapten analogs of the transition states for *p*-nitrophenylphosphorylcholine (Pollack et al., 1986) or for monoaryl phosphonate esters (Tramontano et al., 1986a, 1986b). The artificial monoclonal anti-hapten catalytic Abs were termed abzymes (derived from **a**nti**b**ody en**zyme**).

The evolution of the technology of artificial Abzs during the last two decades has led not only to the rapid development of direct approaches for the generation of Abs with specified properties, but also to the creation of strategies to revise the targeting specificity of individual Abzs. Such modifications of antigen binding specificity can be achieved genetically *in vitro*, by application of the site-directed mutagenesis, or genetic selection or screening (using approaches such as phage display). Alternatively, modification can be induced directly on purified antibody, *via* selective chemical modification by direct introduction of catalytic groups into the Ab combining site. Some studies describing these approaches include (Keinan, 2005, and refs therein). The employment of the approaches have demonstrated that the substrate specificity (and/or the specific activity) of some artificial Abzs is comparable to or even higher than that of enzymes with the same catalytic activity (Barbas et al., 1997; Janda et al., 1997; Keinan, 2005, and refs therein).

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 153

HIV-1 is the etiologic agent of an extremely dangerous human disease, AIDS (Fauci et al., 2008, and refs therein). The association of immune dysfunction in patients with HIV infection and AIDS and the development of AI diseases are very interesting. At this moment the spectrum of reported autoimmune phenomena in AIDS patients is increasing (for review see Zandman-Goddard & Shoenfeld, 2002). A special feature of ADs is high concentrations of auto-Abs (Abs to many different endogenous antigens) (Zouali, 2001; Pisetsky, 2001). The development of AI diseases is characterized by spontaneous generation of primary Abs to proteins, nucleic acids and their complexes, polysaccharides, nucleotides etc. (Earnshaw & Rothfield, 1985; Raptis & Menard, 1980). Later the secondary idiotypic and then antiidiotypic Abs to the primary ones are usually generated, etc. Immunization of animals with DNA or RNA and especially their complexes with proteins leads to the production of

During frank loss of immunocompetence, AI diseases that are predominantly T cell subtype CD8 driven predominate. There is evidence for B cell stimulation and many auto-Abs are reported in HIV patients. HIV-dependent activation of B lymphocytes leads to the production of auto-Abs not only to different viral proteins including HIV reverse transcriptase (RT) and integrase (IN), but also to human cell components, and various immune complexes including anti-cardiolipin, anti-beta2 GPI, anti-DNA, anti-small nuclear ribonucleoproteins, anti-thyroglobulin, anti-thyroid peroxidase, anti-myosin, and antierythropoietin and possibly other human cell and blood components (Fauci et al., 2008; Zandman-Goddard & Shoenfeld, 2002)*.* The list of reported autoimmune diseases in HIV/AIDS includes SLE, anti-phospholipid syndrome, vasculitis, primary biliary cirrhosis, polymyosits, Graves' disease, and idiopathic thrombocytopenic purpura (Zandman-Goddard & Shoenfeld, 2002). The presence of AI phenomena and production of auto-Abs in chronic bacterial and viral infections including HIV could be related to molecular mimicry between microbial or viral and host antigens (Zandman-Goddard & Shoenfeld, 2002; Hentati et al., 1994; Ternynck et al., 1991), altered self, abnormal expression of

**2. Features of the immune status of patients with AIDS, bacterial, and**

anti-DNA and anti-RNA Abs (Gottieb & Shwartz, 1972; Mitsuhashi et al., 1978).

immunoregulatory molecules, and the anti-idiotypic network (Barzilai et al., 2008).

Sato et al., 1994; Hirokawa et al., 2002; Ikeda et al., 2003).

Interestingly, some other viral infections similarly to ADs can also demonstrate AI reactions leading to the formation of Abs to various human and viral antigens. Compared with healthy donors, concentrations of DNA and anti-DNA Abs are higher not only in patients with SLE (36% of SLE patients), but also in multiple sclerosis (17-18%), primary Sjogren's syndrome (18%), Hashimoto's thyroiditis (23%), myasthenia gravis (6%), rheumatoid arthritis (7%), autoimmune hepatitis (Shoenfeld et al., 1988, 1989), and also in lymphoproliferative (Kozyr et al., 1998) and some viral diseases including viral hepatitis, AIDS (Gololobov et al., 1994), and TBE (Garmashova et al., 2004). In the sera of patients with several AI diseases, RNA and anti-RNA Abs were also detected (Blanco et al., 1991;

AIDS, TBE, and hepatitis demonstrating strong reorganization of immune system have some similarities with typical AI diseases such as SLE (HT, and others) which is a systemic AI polyetiologic diffuse disease that is characterized by disorganization of conjunctive tissues with the paramount damage to skin and visceral capillaries (Hhachn, 1996). All known AI and viral diseases like AIDS, viral hepatitis, and TBE are characterized by significant disturb the immune status of the patients accompanied by humoral and cellular

**autoimmune diseases**

Artificial Abzs against transition chemical states of different reactions have been studied intensively (Thayer et al., 1999). Mechanistic basis for the activity of such Abzs is becoming well understood (Janda et al., 1997; Thayer et al., 1999; Keinan, 2005, and refs therein). The field of artificial Abzs has been amply reviewed recently (Martin & Schultz, 1999; Suzuki, 1994; Keinan, 2005, and refs therein), for more detailed description of the relevant reactions.

During last two decades it has become clear that auto-Abs from the sera of patients with different autoimmune (AI) diseases can possess enzymic activities (Suzuki, 1994; Keinan, 2005; Nevinsky et al., 2002a, 2002b, 2003, 2005, 2010a, 2010b, and refs therein). The first example of a natural Abz was an IgG found in bronchial asthma patients, which hydrolyzes intestinal vasoactive peptide (VIP) (Paul et al., 1989), the second was an IgG with DNase activity in SLE (Shuster, et al., 1992), and the third was an IgG with RNase activity in SLE (Buneva et al., 1994). Later, different natural catalytic IgG and/or IgA, IgM hydrolyzing oligopeptides, proteins, DNA, RNA, nucleotides, and polysaccharides were detected in the sera of patients with several autoimmune (AI) and viral pathologies, and Abzs with these and other different activities were discovered in healthy human milk (for review see Nevinsky et al., 2002a, 2002b, 2003, 2005, 2010a, 2010b).

The phenomenon of catalysis by auto-Abzs is extremely interesting and potentially applicable in many different fields including new types of efficient catalysts, new generation of drugs, evaluation of the functional roles of Abzs in innate and adaptive immunity, and understanding of certain aspects of self-tolerance and of the destructive or positive responses in AI diseases. The field of monoclonal Abzs with immunotherapeutic potential has recently been reviewed (Wentworth et al., 1998; Tellier, 2002; Zhou et al., 2002; Nishi, 2003; Stockwin and Holmes, 2003; Hanson et al., 2005; Gabibov et al., 2006; Planque et al., 2008; Wójcik & Kieć-Kononowicz, 2008). Some general possibilities of present and future therapeutic Abs and Abzs application were discussed in (Stockwin & Holmes, 2003). Abs and Abzs can be used to neutralize pathogens, toxins and endogenous mediators of pathology. As cell-targeting reagents, Abs can be used to modulate cytoplasmic cascades or to tag specific cells for complement- or effector-mediated lysis. Abs can also be modified to deliver toxic or modulatory payloads (small molecules, radionuclides and enzymes) and engineered to bind multiple epitopes or even to have novel catalytic activity. The modular structure of Igs and the availability of Ab fragment libraries also make it possible to produce variable-domain therapeutics (Fab, single-chain and domain of Abs). Although exhibiting less favorable kinetics *in vivo*, these fragments are straightforward to express and easily penetrate tissues, making them especially useful as neutralizing or delivery agents. The number of approved Abzs is expected to increase in the near term, as the platform is adopted as a viable alternative to small molecule discovery (Stockwin & Holmes, 2003). The Abzs strategy can be employed for new methods of drug synthesis, as well as for in vivo therapies. Catalytic antibodies seem to be a promising tool for therapeutic purposes, because of their specificity and stereoselectivity. For instance, cocaine-hydrolyzing Abzs have been developed, and may provide a novel approach to the problems of drug addiction (Hanson et al., 2005). Possible application of Abzs for prodrug activation and their potential utility in clinical oncology was also discussed (Nishi, 2003). Abzs have two distinct advantages over canonical enzymes: first, they can be selected to perform reactions not catalyzed by endogenous enzymes, and second, they can be humanized to minimize their immunogenicity (Stockwin & Holmes, 2003).

Artificial Abzs against transition chemical states of different reactions have been studied intensively (Thayer et al., 1999). Mechanistic basis for the activity of such Abzs is becoming well understood (Janda et al., 1997; Thayer et al., 1999; Keinan, 2005, and refs therein). The field of artificial Abzs has been amply reviewed recently (Martin & Schultz, 1999; Suzuki, 1994; Keinan, 2005, and refs therein), for more detailed description of the

During last two decades it has become clear that auto-Abs from the sera of patients with different autoimmune (AI) diseases can possess enzymic activities (Suzuki, 1994; Keinan, 2005; Nevinsky et al., 2002a, 2002b, 2003, 2005, 2010a, 2010b, and refs therein). The first example of a natural Abz was an IgG found in bronchial asthma patients, which hydrolyzes intestinal vasoactive peptide (VIP) (Paul et al., 1989), the second was an IgG with DNase activity in SLE (Shuster, et al., 1992), and the third was an IgG with RNase activity in SLE (Buneva et al., 1994). Later, different natural catalytic IgG and/or IgA, IgM hydrolyzing oligopeptides, proteins, DNA, RNA, nucleotides, and polysaccharides were detected in the sera of patients with several autoimmune (AI) and viral pathologies, and Abzs with these and other different activities were discovered in healthy human milk (for review see

The phenomenon of catalysis by auto-Abzs is extremely interesting and potentially applicable in many different fields including new types of efficient catalysts, new generation of drugs, evaluation of the functional roles of Abzs in innate and adaptive immunity, and understanding of certain aspects of self-tolerance and of the destructive or positive responses in AI diseases. The field of monoclonal Abzs with immunotherapeutic potential has recently been reviewed (Wentworth et al., 1998; Tellier, 2002; Zhou et al., 2002; Nishi, 2003; Stockwin and Holmes, 2003; Hanson et al., 2005; Gabibov et al., 2006; Planque et al., 2008; Wójcik & Kieć-Kononowicz, 2008). Some general possibilities of present and future therapeutic Abs and Abzs application were discussed in (Stockwin & Holmes, 2003). Abs and Abzs can be used to neutralize pathogens, toxins and endogenous mediators of pathology. As cell-targeting reagents, Abs can be used to modulate cytoplasmic cascades or to tag specific cells for complement- or effector-mediated lysis. Abs can also be modified to deliver toxic or modulatory payloads (small molecules, radionuclides and enzymes) and engineered to bind multiple epitopes or even to have novel catalytic activity. The modular structure of Igs and the availability of Ab fragment libraries also make it possible to produce variable-domain therapeutics (Fab, single-chain and domain of Abs). Although exhibiting less favorable kinetics *in vivo*, these fragments are straightforward to express and easily penetrate tissues, making them especially useful as neutralizing or delivery agents. The number of approved Abzs is expected to increase in the near term, as the platform is adopted as a viable alternative to small molecule discovery (Stockwin & Holmes, 2003). The Abzs strategy can be employed for new methods of drug synthesis, as well as for in vivo therapies. Catalytic antibodies seem to be a promising tool for therapeutic purposes, because of their specificity and stereoselectivity. For instance, cocaine-hydrolyzing Abzs have been developed, and may provide a novel approach to the problems of drug addiction (Hanson et al., 2005). Possible application of Abzs for prodrug activation and their potential utility in clinical oncology was also discussed (Nishi, 2003). Abzs have two distinct advantages over canonical enzymes: first, they can be selected to perform reactions not catalyzed by endogenous enzymes, and second, they can be humanized to minimize their

relevant reactions.

Nevinsky et al., 2002a, 2002b, 2003, 2005, 2010a, 2010b).

immunogenicity (Stockwin & Holmes, 2003).

## **2. Features of the immune status of patients with AIDS, bacterial, and autoimmune diseases**

HIV-1 is the etiologic agent of an extremely dangerous human disease, AIDS (Fauci et al., 2008, and refs therein). The association of immune dysfunction in patients with HIV infection and AIDS and the development of AI diseases are very interesting. At this moment the spectrum of reported autoimmune phenomena in AIDS patients is increasing (for review see Zandman-Goddard & Shoenfeld, 2002). A special feature of ADs is high concentrations of auto-Abs (Abs to many different endogenous antigens) (Zouali, 2001; Pisetsky, 2001). The development of AI diseases is characterized by spontaneous generation of primary Abs to proteins, nucleic acids and their complexes, polysaccharides, nucleotides etc. (Earnshaw & Rothfield, 1985; Raptis & Menard, 1980). Later the secondary idiotypic and then antiidiotypic Abs to the primary ones are usually generated, etc. Immunization of animals with DNA or RNA and especially their complexes with proteins leads to the production of anti-DNA and anti-RNA Abs (Gottieb & Shwartz, 1972; Mitsuhashi et al., 1978).

During frank loss of immunocompetence, AI diseases that are predominantly T cell subtype CD8 driven predominate. There is evidence for B cell stimulation and many auto-Abs are reported in HIV patients. HIV-dependent activation of B lymphocytes leads to the production of auto-Abs not only to different viral proteins including HIV reverse transcriptase (RT) and integrase (IN), but also to human cell components, and various immune complexes including anti-cardiolipin, anti-beta2 GPI, anti-DNA, anti-small nuclear ribonucleoproteins, anti-thyroglobulin, anti-thyroid peroxidase, anti-myosin, and antierythropoietin and possibly other human cell and blood components (Fauci et al., 2008; Zandman-Goddard & Shoenfeld, 2002)*.* The list of reported autoimmune diseases in HIV/AIDS includes SLE, anti-phospholipid syndrome, vasculitis, primary biliary cirrhosis, polymyosits, Graves' disease, and idiopathic thrombocytopenic purpura (Zandman-Goddard & Shoenfeld, 2002). The presence of AI phenomena and production of auto-Abs in chronic bacterial and viral infections including HIV could be related to molecular mimicry between microbial or viral and host antigens (Zandman-Goddard & Shoenfeld, 2002; Hentati et al., 1994; Ternynck et al., 1991), altered self, abnormal expression of immunoregulatory molecules, and the anti-idiotypic network (Barzilai et al., 2008).

Interestingly, some other viral infections similarly to ADs can also demonstrate AI reactions leading to the formation of Abs to various human and viral antigens. Compared with healthy donors, concentrations of DNA and anti-DNA Abs are higher not only in patients with SLE (36% of SLE patients), but also in multiple sclerosis (17-18%), primary Sjogren's syndrome (18%), Hashimoto's thyroiditis (23%), myasthenia gravis (6%), rheumatoid arthritis (7%), autoimmune hepatitis (Shoenfeld et al., 1988, 1989), and also in lymphoproliferative (Kozyr et al., 1998) and some viral diseases including viral hepatitis, AIDS (Gololobov et al., 1994), and TBE (Garmashova et al., 2004). In the sera of patients with several AI diseases, RNA and anti-RNA Abs were also detected (Blanco et al., 1991; Sato et al., 1994; Hirokawa et al., 2002; Ikeda et al., 2003).

AIDS, TBE, and hepatitis demonstrating strong reorganization of immune system have some similarities with typical AI diseases such as SLE (HT, and others) which is a systemic AI polyetiologic diffuse disease that is characterized by disorganization of conjunctive tissues with the paramount damage to skin and visceral capillaries (Hhachn, 1996). All known AI and viral diseases like AIDS, viral hepatitis, and TBE are characterized by significant disturb the immune status of the patients accompanied by humoral and cellular

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 155

The origin of natural Abzs in different AI, viral and bacterial diseases may be complex. Similarly to artificial Abzs against analogs of transition states of catalytic reactions, naturally occurring Abzs with DNase and RNase activities may be Abs raised directly against free DNA and RNA or these nucleic acids acting as haptens bound to different proteins and resembling transition states of catalytic reactions (Nevinsky et al., 2003, 2005, 2010a, 2010b, and refs therein). Immunization of rabbits with pure DNA and RNA generated Abs interacting with DNA and possessing weak DNase and RNase activities (Krasnorutskii et al., 2008a, 2008b). Many SLE anti-DNA Abs are directed against histone-DNA nucleosomal complexes appearing as a result of internucleosomal cleavage during apoptosis (Founel & Muller, 2002). Apoptotic cells are the primary source of antigens and immunogens in SLE, and certain features in recognition, processing, and/or presentation of apoptotic autoantigens by antigen-presenting cells can trigger AI processes (Founel & Muller, 2002). Anti-DNA–protein and anti-RNA–protein complexes and other antinuclear antibodies were found in the sera of patients with multisystem connective tissue disease (Gottieb & Shwartz, 1972). Therefore, we have emulated such natural complexes using complexes of DNA and RNA with methylated bovine serum albumin (mBSA). Immunization of rabbits with complex of DNA and RNA with mBSA elicited production of 10-50-fold more active DNase and RNase IgGs, while pIgGs from animals immunized with mBSA were catalytically inactive (Krasnorutskii et al., 2008a, 2008b). Immunization of healthy rabbits with DNase I, DNase II, and pancreatic RNase A also produced anti-idiotypic IgGs with intrinsic DNase and RNase activities (Krasnorutskii et al., 2008c, 2008d, 2009). Thus, DNase and RNase Abzs in different AI diseases may be a cocktail of Abs against complexes of DNA and RNA with proteins and antiidiotypic Abzs to very different DNA- and RNA-

Healthy humans and patients with many diseases with insignificant AI reactions usually lack Abzs or develop Abzs with very low catalytic activities, often on a borderline of the sensitivity of detection methods (Nevinsky et al., 2002a, 2002b, 2003, 2005, 2010a, 2010b, and refs therein).  It was shown, that like in the case of AI-patients, IgGs with DNase activity from autoimmune mice are the earliest and statistically significant markers of pathology and these activities are detectable at the pre-disease stage, when there are no visible markers of SLE pathology or significant proteinuria, and anti-DNA titres are within the typical ranges of these indicators for healthy mice (Dubrovskaya et al., 2003; Andryushkova et al., 2007;

Using different approaches convincing evidence was provided that, similarly to Abzs from SLE and MS patients (Savel'ev et al., 2003; Ivanen et al., 2002, 2004), amylase activity is intrinsic to autoimmune mouse polyclonal IgGs (Andryushkova et al., 2006, 2007). It was shown that the relative activities of IgGs from MRL-lpr/lpr mice in the hydrolysis of DNA, ATP, and polysaccharides correlate very well with some visible (pink spots, baldness of the head and parts of the back, general health deterioration, etc.) and biochemical (proteinuria, Ab titers to native and denatured DNA) markers of AI pathologies during various stages of

hydrolyzing enzymes.

Kuznetsova et al., 2007).

AI reactions, with detectable tissue-specific and organ-nonspecific Abs (Bigazzi, 1983; Sugiama & Yamamota, 1996; Nevinsky, 2010b). At the same time, microbial and viral infections expose the human organism to different components from the parasite's cells and viral particles, including protein, DNA, RNA, lipids, and polysaccharides. The sera of mice infected with different microbe's bacterial pathogens contain a variety of Abs to the parasite's antigens and to human lipids, proteins, and nuclear components, including anti-DNA Abs (Ternynck et al., 1991; Unterkircher et al., 1993; Matsiota-Bernard et al., 1993, 1996; Hentati et al., 1994; Boekel et al., 1997; Wun et al., 2001). The origin of anti-DNA Abs in the infections remains speculative; some of them may arise inadvertently in the course of a normal immune response due to the induction by Abs that bear structures (mimotopes) mimicking DNA (Wun et al., 2001). The immunoregulatory effect of the infection seems to be related, at least partially, to the increase in a particular population of Abs, the polyreactive Abs (Matsiota-Bernard et al., 1996). It has been proposed that bacterial and viral infectious agents can act in some cases through the mechanism of molecular mimicry and stimulate development of different AI diseases. For instance, the agents responsible for molecular mimicry in multiple sclerosis include measles, hepatitis B, herpes simplex, influenza, papilloma, and Epstein-Bar viruses (Steinman, 2001). Thus, AI reactions in different AI, viral and bacterial infection diseases are very similar and may be strongly associated.

#### **3. The origin of artificial and natural abzymes**

Artificial Abzs can be obtained by immunization of animals with chemically stable analogs of transition states of chemical reactions (reviewed in Martin & Schultz, 1999; Nevinsky et al., 2000a; Tanaka, 2002; Tanaka and Barbas, 2002; Dias et al., 2002; Keinan, 2005). On the other hand, artificial antiidiotypic Abs can also possess catalytic activity (Barbas et al., 1997; Wentworth et al., 1998). Building on earlier observations on the existence of idiotypic determinants related to the antigen, Jerne proposed that the immune system is selfregulated by a network of idiotype–anti-idiotype interactions (Jerne, 1974). The simplified model of this network may be schematically presented as follows:

Antibodies 1 and 2 are termed idiotype and anti-idiotype, respectively, etc. There is convincing evidence that such idiotype–anti-idiotype networks are actually present in the body. The presence of blood serum Ab4 (in the notation shown in the scheme) has been confirmed in experimental animals (Jerne, 1974).

If the active site of an enzyme plays the role of antigen triggering this anti-idiotypic chain, it is logical to suggest that the secondary anti-idiotypic Ab2 may possess the structure, a part of which represents an "internal image" or "mould" of the active site of this enzyme, and, consequently, these Abs may possess some properties of this enzyme. This remarkable property of idiotypic mimicry has been exploited to raise monoclonal antiidiotypic Abzs with several different catalytic activities (reviewed in Keinan, 2005; Nevinsky et al., 2005).

AI reactions, with detectable tissue-specific and organ-nonspecific Abs (Bigazzi, 1983; Sugiama & Yamamota, 1996; Nevinsky, 2010b). At the same time, microbial and viral infections expose the human organism to different components from the parasite's cells and viral particles, including protein, DNA, RNA, lipids, and polysaccharides. The sera of mice infected with different microbe's bacterial pathogens contain a variety of Abs to the parasite's antigens and to human lipids, proteins, and nuclear components, including anti-DNA Abs (Ternynck et al., 1991; Unterkircher et al., 1993; Matsiota-Bernard et al., 1993, 1996; Hentati et al., 1994; Boekel et al., 1997; Wun et al., 2001). The origin of anti-DNA Abs in the infections remains speculative; some of them may arise inadvertently in the course of a normal immune response due to the induction by Abs that bear structures (mimotopes) mimicking DNA (Wun et al., 2001). The immunoregulatory effect of the infection seems to be related, at least partially, to the increase in a particular population of Abs, the polyreactive Abs (Matsiota-Bernard et al., 1996). It has been proposed that bacterial and viral infectious agents can act in some cases through the mechanism of molecular mimicry and stimulate development of different AI diseases. For instance, the agents responsible for molecular mimicry in multiple sclerosis include measles, hepatitis B, herpes simplex, influenza, papilloma, and Epstein-Bar viruses (Steinman, 2001). Thus, AI reactions in different AI, viral and bacterial infection diseases are very similar and may be strongly associated.

Artificial Abzs can be obtained by immunization of animals with chemically stable analogs of transition states of chemical reactions (reviewed in Martin & Schultz, 1999; Nevinsky et al., 2000a; Tanaka, 2002; Tanaka and Barbas, 2002; Dias et al., 2002; Keinan, 2005). On the other hand, artificial antiidiotypic Abs can also possess catalytic activity (Barbas et al., 1997; Wentworth et al., 1998). Building on earlier observations on the existence of idiotypic determinants related to the antigen, Jerne proposed that the immune system is selfregulated by a network of idiotype–anti-idiotype interactions (Jerne, 1974). The simplified

Antibodies 1 and 2 are termed idiotype and anti-idiotype, respectively, etc. There is convincing evidence that such idiotype–anti-idiotype networks are actually present in the body. The presence of blood serum Ab4 (in the notation shown in the scheme) has been

If the active site of an enzyme plays the role of antigen triggering this anti-idiotypic chain, it is logical to suggest that the secondary anti-idiotypic Ab2 may possess the structure, a part of which represents an "internal image" or "mould" of the active site of this enzyme, and, consequently, these Abs may possess some properties of this enzyme. This remarkable property of idiotypic mimicry has been exploited to raise monoclonal antiidiotypic Abzs with several different catalytic activities (reviewed in Keinan, 2005; Nevinsky et al., 2005).

**3. The origin of artificial and natural abzymes**

confirmed in experimental animals (Jerne, 1974).

model of this network may be schematically presented as follows:

The origin of natural Abzs in different AI, viral and bacterial diseases may be complex. Similarly to artificial Abzs against analogs of transition states of catalytic reactions, naturally occurring Abzs with DNase and RNase activities may be Abs raised directly against free DNA and RNA or these nucleic acids acting as haptens bound to different proteins and resembling transition states of catalytic reactions (Nevinsky et al., 2003, 2005, 2010a, 2010b, and refs therein). Immunization of rabbits with pure DNA and RNA generated Abs interacting with DNA and possessing weak DNase and RNase activities (Krasnorutskii et al., 2008a, 2008b). Many SLE anti-DNA Abs are directed against histone-DNA nucleosomal complexes appearing as a result of internucleosomal cleavage during apoptosis (Founel & Muller, 2002). Apoptotic cells are the primary source of antigens and immunogens in SLE, and certain features in recognition, processing, and/or presentation of apoptotic autoantigens by antigen-presenting cells can trigger AI processes (Founel & Muller, 2002). Anti-DNA–protein and anti-RNA–protein complexes and other antinuclear antibodies were found in the sera of patients with multisystem connective tissue disease (Gottieb & Shwartz, 1972). Therefore, we have emulated such natural complexes using complexes of DNA and RNA with methylated bovine serum albumin (mBSA). Immunization of rabbits with complex of DNA and RNA with mBSA elicited production of 10-50-fold more active DNase and RNase IgGs, while pIgGs from animals immunized with mBSA were catalytically inactive (Krasnorutskii et al., 2008a, 2008b). Immunization of healthy rabbits with DNase I, DNase II, and pancreatic RNase A also produced anti-idiotypic IgGs with intrinsic DNase and RNase activities (Krasnorutskii et al., 2008c, 2008d, 2009). Thus, DNase and RNase Abzs in different AI diseases may be a cocktail of Abs against complexes of DNA and RNA with proteins and antiidiotypic Abzs to very different DNA- and RNAhydrolyzing enzymes.

Healthy humans and patients with many diseases with insignificant AI reactions usually lack Abzs or develop Abzs with very low catalytic activities, often on a borderline of the sensitivity of detection methods (Nevinsky et al., 2002a, 2002b, 2003, 2005, 2010a, 2010b, and refs therein).  It was shown, that like in the case of AI-patients, IgGs with DNase activity from autoimmune mice are the earliest and statistically significant markers of pathology and these activities are detectable at the pre-disease stage, when there are no visible markers of SLE pathology or significant proteinuria, and anti-DNA titres are within the typical ranges of these indicators for healthy mice (Dubrovskaya et al., 2003; Andryushkova et al., 2007; Kuznetsova et al., 2007).

Using different approaches convincing evidence was provided that, similarly to Abzs from SLE and MS patients (Savel'ev et al., 2003; Ivanen et al., 2002, 2004), amylase activity is intrinsic to autoimmune mouse polyclonal IgGs (Andryushkova et al., 2006, 2007). It was shown that the relative activities of IgGs from MRL-lpr/lpr mice in the hydrolysis of DNA, ATP, and polysaccharides correlate very well with some visible (pink spots, baldness of the head and parts of the back, general health deterioration, etc.) and biochemical (proteinuria, Ab titers to native and denatured DNA) markers of AI pathologies during various stages of

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 157

proteinuria, but also with a significant additional change in the profile of HSC differentiation. This change seems to be the most important factor in the irreversible switching of the mouse immune system to an AI mode, since the changes in cell proliferation and apoptosis in different organs occur mainly on transition from healthy to pre-diseased mice and the observed differences in these indices between pre-diseased and diseased mice are insignificant (Andryushkova et al., 2006, 2007, 2009). Immunization of healthy young AI mice leads to the highest increase in urine protein, titers of anti-DNA Abs as well as DNase, amylase and ATPase Abz activities, occurring in parallel with a significant decrease in apoptosis, especially in bone marrow, thymus and spleen. However, the profile of HSC differentiation in immunized mice is quite different from the pre-diseased and spontaneously diseased mice, but comparable with that for young healthy animals. It was shown, that in contrast to spontaneously diseased AI mice, immunization with DNA does not remarkably affect bone marrow stem cells; the increased levels of anti-DNA Abs and Abzs in immunized mice may be mainly provided by an activation of lymphocyte differentiation and proliferation in different organs, first of all in the spleen, with a concomitant decrease in apoptosis. A significant decrease in apoptosis in the immunized mice may be an important factor providing the increased number of specific lymphocytes producing auto-Abs and Abzs, which are normally eliminated. Very high urine protein concentration and visible markers of SLE demonstrated by the immunized mice may be a result of kidney and spleen dysfunction (Andryushkova et al., 2006, 2007, 2009). Overall, in contrast to immunization of healthy mice an appearance of Abzs and increase in their activity is associated with changes in differentiation and proliferation of mice bone marrow HSC. At the onset of AI diseases (pre-disease condition), Abs are usually contain catalytic Abzs produced by a single clone, or at least a relatively narrow repertoire of Abzs with relatively low relative activities. In the course of chronic AI pathology development, the repertoire of Abzs constantly widens and Abs with significantly higher RAs can be found. In addition, the number of Abzs with high RAs usually increases during exacerbation of AI pathologies (Nevinsky et al., 2003, 2005, 2010a, 2010b, and refs therein). It should be mentioned, that the detection of Abzs was shown to be the earliest and statistically significant indicator of development of different autoimmune diseases in humans (Nevinsky et al., 2003, 2005, 2010a, 2010b, and refs therein)

Natural Abzs from sera of patients are usually polyclonal in origin and are products of different immuno-competent cells (Nevinsky et al., 2000b, 2002a, 2002b, 2003, 2005, 2010a, 2010b, and refs therein). Natural Abz purification is one of the most complicated aspects of

In study Abzs from the sera of HIV-infected patients, electrophoretically and immunologically homogeneous AIDS Ab fraction (pIgG+pIgM+pIgA) was first purified by chromatography of the serum proteins on Protein A-Sepharose under conditions that remove non-specifically bound proteins (Odintsova et al., 2006a, 2006b; Baranova et al., 2009, 2010). pIgMs were separated from pIgAs and pIgGs by FPLC gel filtration of the total Ab fraction on a Superdex 200 (Baranova et al., 2009, 2010). The homogeneity of the 150 kDa IgG was confirmed by SDS-PAGE with silver staining, which showed a single band under nonreducing conditions and two bands corresponding to the H and L chains after reduction

and animals (Andryushkova et al., 2006, 2007, 2009).

**4.1 Purification of natural abzymes**

**4. Catalytic antibodies of HIV-infected patients**

their study; it was discussed in detail in review (Nevinsky et al., 2000b).

mouse SLE (Andryushkova et al., 2006, 2007, 2009). Similarly to Abzs with DNase and RNase activities, catalytic Abs with polysaccharide-hydrolysing activity can be Abs directly against polysaccharides and their complexes with proteins and enzymes and second antiidiotypic Igs against different enzymes hydrolyzing polysaccharides (Andryushkova et al., 2006, 2007, 2009; Nevinsky et al., 2005, 2010a, 2010b).

IgGs and/or IgMs and IgAs hydrolyzing different peptides and proteins were also found in AI and other diseases: vasoactive intestinal peptide (VIP) in asthma (Paul et al., 1989), thyroglobulin in HT and rheumatoid arthritis (Li et al., 1995; Kalaga et al., 1995), prothrombin in multiple myeloma (Thiagarajan et al., 2000), protein factor VIII in haemophilia A (Lacroix-Desmazes et al., 1999), and myelin basic protein (MBP) in MS (Polosukhina et al., 2004, 2005, 2006, 2006; Legostaeva et al., 2010). Some healthy humans produce Abzs with low VIP- (Paul et al., 1989), and thyroglobulin-hydrolyzing activities (Kalaga et al., 1995), but usually healthy volunteers and patients with many diseases with insignificant autoimmune reactions lack Abzs with proteolytic activity (Nevinsky et al., 2002a, 2002b, 2003, 2005, 2010a, 2010b, and refs therein). Since immunization of AI mice results in a dramatically higher incidence of Abzs with a higher activity than in conventionally used normal mouse strains (Tawfik et al., 2002; Nishi, 2002), the formation of Abzs in AI and some viral diseases may be much more profuse. The question is why autoimmunization of AI patients and mice results in a dramatically higher incidence of catalytically inactive Abs and Abzs with enzyme properties as compared with healthy humans and animals.

MRL-lpr/lpr mice spontaneously developing a SLE-like disorder are a very promising model to study the mechanisms of natural Abzs generation and their role in the pathogenesis of pronounced AI disturbances. A mutation in the *lpr* gene of these mice leads to a deficit in functional Fas ligand and dysregulation of apoptosis in homozygotes (Watanabe-Fukunada et al., 1992; Nagata & Suda, 1995). As a result, the mice develop SLElike phenotype, including accumulation of double-negative T cells (CD4– CD8– B220+ TCR+) in the peripheral lymphoid organs.

Recently we have carried out the first analysis of possible correlations between the relative activities (RAs) of mouse IgGs in the hydrolysis of DNA, ATP, and polysaccharides with several clinical and biochemical markers of AI pathologies (proteinuria, Ab titers to native and denatured DNA) at various stages of mouse SLE (Andryushkova et al., 2006, 2007, 2009). An ever-growing number of observations suggested that AI diseases may originate from defects in hematopoietic stem cells (Ikehara et al., 1990). Therefore, lymphocyte proliferation and apoptosis at different stages of the AI disorder development in MRL-lpr/lpr mice were also studied. It was shown that IgGs from the sera of 2-7 month-old control non-autoimmune (CBAxC57BL)F1 and BALB/c mice and 2-3 months-old MRL-lpr/lpr mice (conditionally healthy mice) are catalytically inactive (Andryushkova et al., 2006, 2007, 2009). During spontaneous development of deep SLE-like pathology a specific reorganization of immune system of these mice leads to conditions associated with a production of IgGs hydrolyzing DNA, ATP, and polysaccharides with low catalytic activities (conditionally pre-diseased mice) (Andryushkova et al., 2006, 2007, 2009). First significant changes in differentiation and proliferation of mice bone marrow hematopoietic stem cells (HSC; granulocyticmacrophagic colony-forming unit; erythroid burst-forming unit, and granulocyticerythroid-megacaryocytic-macrophagic colony-forming unit) in pre-diseased in comparison with healthy mice are most likely only temporary, since a transition from the pre-diseased to diseased mice is associated not only with an increase in the RAs of different Abzs and

mouse SLE (Andryushkova et al., 2006, 2007, 2009). Similarly to Abzs with DNase and RNase activities, catalytic Abs with polysaccharide-hydrolysing activity can be Abs directly against polysaccharides and their complexes with proteins and enzymes and second antiidiotypic Igs against different enzymes hydrolyzing polysaccharides (Andryushkova et

IgGs and/or IgMs and IgAs hydrolyzing different peptides and proteins were also found in AI and other diseases: vasoactive intestinal peptide (VIP) in asthma (Paul et al., 1989), thyroglobulin in HT and rheumatoid arthritis (Li et al., 1995; Kalaga et al., 1995), prothrombin in multiple myeloma (Thiagarajan et al., 2000), protein factor VIII in haemophilia A (Lacroix-Desmazes et al., 1999), and myelin basic protein (MBP) in MS (Polosukhina et al., 2004, 2005, 2006, 2006; Legostaeva et al., 2010). Some healthy humans produce Abzs with low VIP- (Paul et al., 1989), and thyroglobulin-hydrolyzing activities (Kalaga et al., 1995), but usually healthy volunteers and patients with many diseases with insignificant autoimmune reactions lack Abzs with proteolytic activity (Nevinsky et al., 2002a, 2002b, 2003, 2005, 2010a, 2010b, and refs therein). Since immunization of AI mice results in a dramatically higher incidence of Abzs with a higher activity than in conventionally used normal mouse strains (Tawfik et al., 2002; Nishi, 2002), the formation of Abzs in AI and some viral diseases may be much more profuse. The question is why autoimmunization of AI patients and mice results in a dramatically higher incidence of catalytically inactive Abs and Abzs with enzyme properties as compared with healthy

MRL-lpr/lpr mice spontaneously developing a SLE-like disorder are a very promising model to study the mechanisms of natural Abzs generation and their role in the pathogenesis of pronounced AI disturbances. A mutation in the *lpr* gene of these mice leads to a deficit in functional Fas ligand and dysregulation of apoptosis in homozygotes (Watanabe-Fukunada et al., 1992; Nagata & Suda, 1995). As a result, the mice develop SLElike phenotype, including accumulation of double-negative T cells (CD4– CD8– B220+ TCR+)

Recently we have carried out the first analysis of possible correlations between the relative activities (RAs) of mouse IgGs in the hydrolysis of DNA, ATP, and polysaccharides with several clinical and biochemical markers of AI pathologies (proteinuria, Ab titers to native and denatured DNA) at various stages of mouse SLE (Andryushkova et al., 2006, 2007, 2009). An ever-growing number of observations suggested that AI diseases may originate from defects in hematopoietic stem cells (Ikehara et al., 1990). Therefore, lymphocyte proliferation and apoptosis at different stages of the AI disorder development in MRL-lpr/lpr mice were also studied. It was shown that IgGs from the sera of 2-7 month-old control non-autoimmune (CBAxC57BL)F1 and BALB/c mice and 2-3 months-old MRL-lpr/lpr mice (conditionally healthy mice) are catalytically inactive (Andryushkova et al., 2006, 2007, 2009). During spontaneous development of deep SLE-like pathology a specific reorganization of immune system of these mice leads to conditions associated with a production of IgGs hydrolyzing DNA, ATP, and polysaccharides with low catalytic activities (conditionally pre-diseased mice) (Andryushkova et al., 2006, 2007, 2009). First significant changes in differentiation and proliferation of mice bone marrow hematopoietic stem cells (HSC; granulocyticmacrophagic colony-forming unit; erythroid burst-forming unit, and granulocyticerythroid-megacaryocytic-macrophagic colony-forming unit) in pre-diseased in comparison with healthy mice are most likely only temporary, since a transition from the pre-diseased to diseased mice is associated not only with an increase in the RAs of different Abzs and

al., 2006, 2007, 2009; Nevinsky et al., 2005, 2010a, 2010b).

humans and animals.

in the peripheral lymphoid organs.

proteinuria, but also with a significant additional change in the profile of HSC differentiation. This change seems to be the most important factor in the irreversible switching of the mouse immune system to an AI mode, since the changes in cell proliferation and apoptosis in different organs occur mainly on transition from healthy to pre-diseased mice and the observed differences in these indices between pre-diseased and diseased mice are insignificant (Andryushkova et al., 2006, 2007, 2009). Immunization of healthy young AI mice leads to the highest increase in urine protein, titers of anti-DNA Abs as well as DNase, amylase and ATPase Abz activities, occurring in parallel with a significant decrease in apoptosis, especially in bone marrow, thymus and spleen. However, the profile of HSC differentiation in immunized mice is quite different from the pre-diseased and spontaneously diseased mice, but comparable with that for young healthy animals. It was shown, that in contrast to spontaneously diseased AI mice, immunization with DNA does not remarkably affect bone marrow stem cells; the increased levels of anti-DNA Abs and Abzs in immunized mice may be mainly provided by an activation of lymphocyte differentiation and proliferation in different organs, first of all in the spleen, with a concomitant decrease in apoptosis. A significant decrease in apoptosis in the immunized mice may be an important factor providing the increased number of specific lymphocytes producing auto-Abs and Abzs, which are normally eliminated. Very high urine protein concentration and visible markers of SLE demonstrated by the immunized mice may be a result of kidney and spleen dysfunction (Andryushkova et al., 2006, 2007, 2009). Overall, in contrast to immunization of healthy mice an appearance of Abzs and increase in their activity is associated with changes in differentiation and proliferation of mice bone marrow HSC. At the onset of AI diseases (pre-disease condition), Abs are usually contain catalytic Abzs produced by a single clone, or at least a relatively narrow repertoire of Abzs with relatively low relative activities. In the course of chronic AI pathology development, the repertoire of Abzs constantly widens and Abs with significantly higher RAs can be found. In addition, the number of Abzs with high RAs usually increases during exacerbation of AI pathologies (Nevinsky et al., 2003, 2005, 2010a, 2010b, and refs therein). It should be mentioned, that the detection of Abzs was shown to be the earliest and statistically significant indicator of development of different autoimmune diseases in humans (Nevinsky et al., 2003, 2005, 2010a, 2010b, and refs therein) and animals (Andryushkova et al., 2006, 2007, 2009).

## **4. Catalytic antibodies of HIV-infected patients**

#### **4.1 Purification of natural abzymes**

Natural Abzs from sera of patients are usually polyclonal in origin and are products of different immuno-competent cells (Nevinsky et al., 2000b, 2002a, 2002b, 2003, 2005, 2010a, 2010b, and refs therein). Natural Abz purification is one of the most complicated aspects of their study; it was discussed in detail in review (Nevinsky et al., 2000b).

In study Abzs from the sera of HIV-infected patients, electrophoretically and immunologically homogeneous AIDS Ab fraction (pIgG+pIgM+pIgA) was first purified by chromatography of the serum proteins on Protein A-Sepharose under conditions that remove non-specifically bound proteins (Odintsova et al., 2006a, 2006b; Baranova et al., 2009, 2010). pIgMs were separated from pIgAs and pIgGs by FPLC gel filtration of the total Ab fraction on a Superdex 200 (Baranova et al., 2009, 2010). The homogeneity of the 150 kDa IgG was confirmed by SDS-PAGE with silver staining, which showed a single band under nonreducing conditions and two bands corresponding to the H and L chains after reduction

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 159

a typical example for AIDS IgGs (lane 1); there was no hydrolysis of DNA by control Abs from healthy donors (lane 2). Control human urine (lane 3) and bovine pancreatic DNase I (lane 4) also cleaved DNA, but produced bands in the position well below (33–36 kDa) the intact pIgGs (150 kDa). Since SDS dissociates all protein complexes, the detection of the DNase activity in the gel zone corresponding only to intact IgGs together with the absence of any other activity band or protein band (Fig. 2B), provides direct evidence that AIDS pIgGs hydrolyze DNA and are not contaminated by canonical DNases. In addition, after incubation of IgGs with DTT only light chains of AIDS IgGs demonstrated DNase activity

**A B C**

Fig. 2. *In situ* SDS-PAGE analysis of DNase activity of AIDS intact IgGs (lane 1), healthy humans (lane 2) (B); human urine (lane 3) and bovine pancreatic DNase I (lane 4, B) in nonreducing conditions. IgGs were analyzed in reducing conditions after Abs incubation with DTT (C). After electrophoresis, the gel containing 3 g/ml thymus DNA (B and C) was incubated under special conditions for protein refolding and hydrolysis of nucleic acids; the nuclease activity was visualized by ethidium bromide staining of the gels (B and C). The longitudinal slices of the same gel were used for Coomassie R250 staining to reveal the positions of IgG bands (lane 2, A) and protein molecular mass markers (lane 1, A).

AIDS IgGs and IgMs were separated by SDS-PAGE respectively under nonreducing and reducing conditions and their proteolytic activity was detected after the extraction of proteins from excised gel slices (Baranova et. al., 2009, 2010). The detection of INhydrolyzing activity in the gel region corresponding only to IgG, together with the absence of any other bands of the activity or protein, provided direct evidence that IgG possesses INhydrolyzing activity. Similar results were obtained for AIDS IgGs hydrolyzing HIV RT,

As mentioned above, pIgMs cannot enter the gel. The absence of IN-hydrolyzing activity from all gel zones corresponding to the intact pIgMs under nonreducing conditions (data not shown), together with hydrolysis of IN only with separated heavy and light chains of IgMs under reducing conditions and the absence of any other bands of the activity (Fig. 3)

It was shown (Odintsova et. al., 2006b), that in contrast to known different nonspecific proteases hydrolyzing many proteins, AIDS pIgGs non-fractionated by affinity chromatography on Sepharoses bearing specific immobilized proteins efficiently hydrolyze only: human casein > HIV-1 RT > HSA but not many other tested proteins. Later it was shown that AIDS IgGs and IgMs also hydrolyze HIV-1 integrase (for example, Fig. 4)

provides a direct evidence that IgM possesses IN-hydrolyzing activity.

**IgG**

**H chain**

**L chain**

**1 2 1 2 3 4**

(Fig. 2C).

**kDa**

**150**

**14**

HSA, and human casein (Odintsova et. al., 2006b).

(Baranova et. al., 2009, 2010).

(Fig. 1). Since IgM has a very high molecular mass (~970 kDa), it cannot enter SDS-PAGE gels under nonreducing conditions (Fig. 1A, lane 2). Two bands corresponding to the H and L chains of pIgMs were evident after Ab reduction (Fig. 1B, lane 1). The absence of any protein bands in the gel corresponding to pIgMs under nonreducing conditions (Fig. 1A, lane 2) and the presence of only two bands corresponding to the heavy and light chains under reducing conditions (Fig. 1B, lane 1) demonstrates the absence of protein contaminations in the pIgM preparations.

Fig. 1. SDS-PAGE in a nonreducing 4–15% gradient gel followed by silver staining of control pIgGs (lane 1) and pIgMs (lane 2) after affinity chromatography on protein A-Sepharose and FPLC gel filtration on a Superdex 200 column (A). SDS-PAGE of pIgGs (lane 2) and pIgMs (lane 1) in a reducing 12% gel (B).

#### **4.2 Criteria to establish that catalytic activity is intrinsic to antibodies**

The application of rigid criteria allowed the authors of the first article concerning natural Abzs (Paul et al., 1989) to conclude that VIP-hydrolyzing activity is an intrinsic property of Abs from the sera of patients with asthma. Later several additional criteria were proposed (for review see Nevinsky et al., 2000a, 2002a, 2002b, 2003, 2005).

It was shown that non-fractionated on affinity sorbents bearing immobilized DNA or specific protein substrates, pIgGs and pIgMs from HIV infected patients effectively hydrolyze DNA (Odintsova et. al., 2006a), HIV-1 RT, human casein, human serum albumin (HAS; Odintsova et. al., 2006b), and HIV-1 IN (Baranova et. al., 2009, 2010) but not many other tested proteins. We applied a set of strict criteria worked out previously (Paul et al., 1989, Nevinsky et al., 2000a, 2002a) for an analysis of DNase and proteolytic activity as an intrinsic property of AIDS IgGs and/or IgMs. The most important of these are: i) electrophoretic homogeneity of pIgGs and pIgMs (Fig. 1); ii) the complete absorption of AIDS IgGs and IgMs with the DNase or proteolytic activities by Sepharose bearing immobilized anti-light chain of human Abs leading to a disappearance of the activity from the solution and recover following its elution with an acidic buffer (pH 2.6); iii) FPLC gel filtration of IgGs using an acidic buffer (pH 2.6) did not lead to a disappearance of the activity, which tracked exactly with IgGs or IgMs. The fulfilment of these criteria was observed for Abzs with all activities mentioned above (Odintsova et. al., 2006a, 2006b; Baranova et. al., 2009, 2010).

To exclude possible artefacts due to hypothetical traces of contaminating enzymes, pIgGs were subjected to SDS-PAGE in a gel co-polymerized with calf thymus DNA, and their DNase activity was detected by incubating the gel in the standard reaction buffer (Fig. 2). Ethidium bromide staining of the gels after the electrophoresis and refolding of IgGs revealed sharp dark bands against a fluorescent background of DNA. Fig. 2B demonstrates

(Fig. 1). Since IgM has a very high molecular mass (~970 kDa), it cannot enter SDS-PAGE gels under nonreducing conditions (Fig. 1A, lane 2). Two bands corresponding to the H and L chains of pIgMs were evident after Ab reduction (Fig. 1B, lane 1). The absence of any protein bands in the gel corresponding to pIgMs under nonreducing conditions (Fig. 1A, lane 2) and the presence of only two bands corresponding to the heavy and light chains under reducing conditions (Fig. 1B, lane 1) demonstrates the absence of protein

> **H(IgM) H(IgG)**

> > **L**

**42**

**67 94 kDa**

> **29 14**

**42 94 150**

**A B**

Fig. 1. SDS-PAGE in a nonreducing 4–15% gradient gel followed by silver staining of control pIgGs (lane 1) and pIgMs (lane 2) after affinity chromatography on protein A-Sepharose and FPLC gel filtration on a Superdex 200 column (A). SDS-PAGE of pIgGs (lane 2) and pIgMs

The application of rigid criteria allowed the authors of the first article concerning natural Abzs (Paul et al., 1989) to conclude that VIP-hydrolyzing activity is an intrinsic property of Abs from the sera of patients with asthma. Later several additional criteria were proposed

It was shown that non-fractionated on affinity sorbents bearing immobilized DNA or specific protein substrates, pIgGs and pIgMs from HIV infected patients effectively hydrolyze DNA (Odintsova et. al., 2006a), HIV-1 RT, human casein, human serum albumin (HAS; Odintsova et. al., 2006b), and HIV-1 IN (Baranova et. al., 2009, 2010) but not many other tested proteins. We applied a set of strict criteria worked out previously (Paul et al., 1989, Nevinsky et al., 2000a, 2002a) for an analysis of DNase and proteolytic activity as an intrinsic property of AIDS IgGs and/or IgMs. The most important of these are: i) electrophoretic homogeneity of pIgGs and pIgMs (Fig. 1); ii) the complete absorption of AIDS IgGs and IgMs with the DNase or proteolytic activities by Sepharose bearing immobilized anti-light chain of human Abs leading to a disappearance of the activity from the solution and recover following its elution with an acidic buffer (pH 2.6); iii) FPLC gel filtration of IgGs using an acidic buffer (pH 2.6) did not lead to a disappearance of the activity, which tracked exactly with IgGs or IgMs. The fulfilment of these criteria was observed for Abzs with all activities mentioned above (Odintsova et. al., 2006a, 2006b;

To exclude possible artefacts due to hypothetical traces of contaminating enzymes, pIgGs were subjected to SDS-PAGE in a gel co-polymerized with calf thymus DNA, and their DNase activity was detected by incubating the gel in the standard reaction buffer (Fig. 2). Ethidium bromide staining of the gels after the electrophoresis and refolding of IgGs revealed sharp dark bands against a fluorescent background of DNA. Fig. 2B demonstrates

**IgM**

**IgG**

**4.2 Criteria to establish that catalytic activity is intrinsic to antibodies**

(for review see Nevinsky et al., 2000a, 2002a, 2002b, 2003, 2005).

contaminations in the pIgM preparations.

(lane 1) in a reducing 12% gel (B).

Baranova et. al., 2009, 2010).

a typical example for AIDS IgGs (lane 1); there was no hydrolysis of DNA by control Abs from healthy donors (lane 2). Control human urine (lane 3) and bovine pancreatic DNase I (lane 4) also cleaved DNA, but produced bands in the position well below (33–36 kDa) the intact pIgGs (150 kDa). Since SDS dissociates all protein complexes, the detection of the DNase activity in the gel zone corresponding only to intact IgGs together with the absence of any other activity band or protein band (Fig. 2B), provides direct evidence that AIDS pIgGs hydrolyze DNA and are not contaminated by canonical DNases. In addition, after incubation of IgGs with DTT only light chains of AIDS IgGs demonstrated DNase activity (Fig. 2C).

Fig. 2. *In situ* SDS-PAGE analysis of DNase activity of AIDS intact IgGs (lane 1), healthy humans (lane 2) (B); human urine (lane 3) and bovine pancreatic DNase I (lane 4, B) in nonreducing conditions. IgGs were analyzed in reducing conditions after Abs incubation with DTT (C). After electrophoresis, the gel containing 3 g/ml thymus DNA (B and C) was incubated under special conditions for protein refolding and hydrolysis of nucleic acids; the nuclease activity was visualized by ethidium bromide staining of the gels (B and C). The longitudinal slices of the same gel were used for Coomassie R250 staining to reveal the positions of IgG bands (lane 2, A) and protein molecular mass markers (lane 1, A).

AIDS IgGs and IgMs were separated by SDS-PAGE respectively under nonreducing and reducing conditions and their proteolytic activity was detected after the extraction of proteins from excised gel slices (Baranova et. al., 2009, 2010). The detection of INhydrolyzing activity in the gel region corresponding only to IgG, together with the absence of any other bands of the activity or protein, provided direct evidence that IgG possesses INhydrolyzing activity. Similar results were obtained for AIDS IgGs hydrolyzing HIV RT, HSA, and human casein (Odintsova et. al., 2006b).

As mentioned above, pIgMs cannot enter the gel. The absence of IN-hydrolyzing activity from all gel zones corresponding to the intact pIgMs under nonreducing conditions (data not shown), together with hydrolysis of IN only with separated heavy and light chains of IgMs under reducing conditions and the absence of any other bands of the activity (Fig. 3) provides a direct evidence that IgM possesses IN-hydrolyzing activity.

It was shown (Odintsova et. al., 2006b), that in contrast to known different nonspecific proteases hydrolyzing many proteins, AIDS pIgGs non-fractionated by affinity chromatography on Sepharoses bearing specific immobilized proteins efficiently hydrolyze only: human casein > HIV-1 RT > HSA but not many other tested proteins. Later it was shown that AIDS IgGs and IgMs also hydrolyze HIV-1 integrase (for example, Fig. 4) (Baranova et. al., 2009, 2010).

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 161

 (Odintsova et al., 2006b). It means that within pools of AIDs pIgGs and pIgMs only specific anti-IN Abzs are able to hydrolyze intact globular molecules of viral integrase (Baranoba et al., 2009, 2010), while specific anti-HSA, anti-casein, and anti-RT IgGs hydrolyze their

**4.3 Comparison of the relative catalytic activity of Abs from different AIDS patients**  Sera of ten healthy donors, 110 HIV-infected patients (18–40-years-old; men and women) including 65 at the stage of pre-AIDS and 45 at the stage of generalized lymphadenopathy (GL) according to the classification of the Center of Disease Control and Prevention were used to analyze the catalytic activities of IgGs and IgMs (Odintsova et al., 2006a, 2006b; Baranova et al., 2009, 2010). Patients with pre-AIDS stage were characterized by a decrease in their body mass up to 10%, fungal, bacterial and viral lesions of skin and mucosal surfaces, shingles, repeating pharyngitis, sinusitis, otitis, and frequent acute respiratory infection. Polyclonal IgGs from 10 healthy controls were inactive in DNA hydrolysis (Odintsova et al., 2006a). Similar results were obtained earlier for several groups of 10-20 healthy humans used as controls in the studies of DNase Abs from the sera of patients with AI diseases (Nevinsky *et al.,* 2003, 2005, 2010a, 2010b and refs therein).The type of plasmid supercoiled (sc) DNA hydrolysis by AIDS pIgGs did not depend on the Ab concentration and the rate of the hydrolysis linearly increased with the increase in IgG concentration and time of incubation (Odintsova et al., 2006a). Fig. 5 illustrates a cleavage of plasmid DNA (14 g/ml) by Abs (0.3 mg/ml) from several AIDS patients after 4 h of incubation. One can see that in this period some Abs cause only single breaks in one strand of supercoiled DNA (lanes 1-3), whereas others cause multiple breaks and as a result the formation of linear DNA (lanes 4-6). The most active Abs hydrolyze DNA into short and medium length ODNs (lanes 7-10). It should be mentioned that Fig. 5 in principle illustrates a range of possible changes of the relative DNase activities for patients with not only AIDS but also with different AI diseases and viral pathologies previously analyzed (Nevinsky *et al.,* 2000a, 2003, 2005, 2010a, 2010b and refs therein). When passing from one pathology to another only the values of a relative

percent of patients with low, middle and high DNase activities is usually changed.

Fig. 5. DNase activities of catalytic AIDS IgGs from different patients in the cleavage of supercoiled (sc) and nicked (n) plasmid DNA (14 g/ml). Lanes 1–10, IgGs (0.3 mg/ml; incubation for 4 h) from the sera of 10 different patients; C1, scDNA incubated alone; C2 and

The efficiency of DNA cleavage was calculated from the increase of DNA in the band of scDNA (10-40 % of initial DNA hydrolysis); the measured RAs (%) for IgGs were normalized to standard conditions (0.1 mg/ml Abs, 2 h) and a complete transition of scDNA to its relaxed form was taken for 100%. While four AIDS IgG preparations were completely inactive, the remaining 106 IgG samples (96 %) demonstrated the RAs from 5 to 100% of

C3, scDNA incubated with Ab from the sera of two healthy donors.

scDNA hydrolysis (Table 1).

specific target proteins (Odintsova et. al., 2006b).

Fig. 3. SDS-PAGE analysis of IN-hydrolyzing activity of AIDS IgMs. After reducing SDS-PAGE of purified AIDS IgMs the gel was incubated under special conditions for renaturation of Abs. The relative IN-hydrolyzing activity (RA, %) was quantified using the extracts of fragments (2–3-mm each) of one longitudinal slice of the gel corresponding to 3 individual IgMs: (■), IgM1; (□), IgM2; (∆), IgM3.

It was shown that immunogenic VIP (Paul et al., 1989), human MBP (Ponomarenko et al., 2006; Legostaseva at al., 2010), human milk casein (Odintsova et al., 2011), stimulate formation of Abzs which in contrast to canonical proteases efficiently hydrolyze only antigen-proteins, but not many other proteins tested. To analyze the "average" proteolytic activity of AIDS Abzs, two mixtures of equal amounts of electrophoretically homogeneous pIgGs (pIgGmix) and pIgMs (pIgMmix) with different relatively high and average activities from the sera of seven patients were prepared. After purification of anti-IN Abs on IN-Sepharose these Abzs hydrolyzed only IN and cannot hydrolyze other proteins including viral RT (Fig. 4). In addition, it was shown that AIDS pIgGs and pIgMs after their purification on sorbents bearing immobilized HIV RT (Fig. 4), human casein or HSA specifically hydrolyzed only cognate protein, but not many other proteins including HIV IN

Fig. 4. SDS-PAGE analysis of protein hydrolysis by AIDS IgGs and IgMs. A, HIV-1 IN was incubated for alone (1) or in the presence of pIgGs from two healthy donors (2 and 3) and pIgGs from two different AIDS patients (4 and 5), or with AIDS IgGs purified on IN-Sepharose (6). B, AIDS IgMs purified on RT-Sepharose were incubated with different proteins without (odd numbers) and with Abs (even numbers): HIV-1 IN (1 and 2); p66 HIV RT (3 and 4); HSA (5 and 6); myelin basic protein (7 and 8). Lane 9 corresponds to mixture of standard protein markers with known molecular masses.

**205**

**kDa**

individual IgMs: (■), IgM1; (□), IgM2; (∆), IgM3.

**RA, %**

**150**

PAGE of purified AIDS IgMs the gel was incubated under special conditions for

**97**

Fig. 3. SDS-PAGE analysis of IN-hydrolyzing activity of AIDS IgMs. After reducing SDS-

renaturation of Abs. The relative IN-hydrolyzing activity (RA, %) was quantified using the extracts of fragments (2–3-mm each) of one longitudinal slice of the gel corresponding to 3

It was shown that immunogenic VIP (Paul et al., 1989), human MBP (Ponomarenko et al., 2006; Legostaseva at al., 2010), human milk casein (Odintsova et al., 2011), stimulate formation of Abzs which in contrast to canonical proteases efficiently hydrolyze only antigen-proteins, but not many other proteins tested. To analyze the "average" proteolytic activity of AIDS Abzs, two mixtures of equal amounts of electrophoretically homogeneous pIgGs (pIgGmix) and pIgMs (pIgMmix) with different relatively high and average activities from the sera of seven patients were prepared. After purification of anti-IN Abs on IN-Sepharose these Abzs hydrolyzed only IN and cannot hydrolyze other proteins including viral RT (Fig. 4). In addition, it was shown that AIDS pIgGs and pIgMs after their purification on sorbents bearing immobilized HIV RT (Fig. 4), human casein or HSA specifically hydrolyzed only cognate protein, but not many other proteins including HIV IN

**1 2 3 4 5 6 1 2 3 4 5 6 7 8 9**

**kDa kDa**

**67 43 30**

**14.4**

Fig. 4. SDS-PAGE analysis of protein hydrolysis by AIDS IgGs and IgMs. A, HIV-1 IN was incubated for alone (1) or in the presence of pIgGs from two healthy donors (2 and 3) and pIgGs from two different AIDS patients (4 and 5), or with AIDS IgGs purified on IN-Sepharose (6). B, AIDS IgMs purified on RT-Sepharose were incubated with different proteins without (odd numbers) and with Abs (even numbers): HIV-1 IN (1 and 2); p66 HIV RT (3 and 4); HSA (5 and 6); myelin basic protein (7 and 8). Lane 9 corresponds to mixture of

**20**

standard protein markers with known molecular masses.

**A B**

**43**

**0 3 6 9 12 15 18 21 24**

**Fragment number**

**30**

**18**

 (Odintsova et al., 2006b). It means that within pools of AIDs pIgGs and pIgMs only specific anti-IN Abzs are able to hydrolyze intact globular molecules of viral integrase (Baranoba et al., 2009, 2010), while specific anti-HSA, anti-casein, and anti-RT IgGs hydrolyze their specific target proteins (Odintsova et. al., 2006b).

## **4.3 Comparison of the relative catalytic activity of Abs from different AIDS patients**

Sera of ten healthy donors, 110 HIV-infected patients (18–40-years-old; men and women) including 65 at the stage of pre-AIDS and 45 at the stage of generalized lymphadenopathy (GL) according to the classification of the Center of Disease Control and Prevention were used to analyze the catalytic activities of IgGs and IgMs (Odintsova et al., 2006a, 2006b; Baranova et al., 2009, 2010). Patients with pre-AIDS stage were characterized by a decrease in their body mass up to 10%, fungal, bacterial and viral lesions of skin and mucosal surfaces, shingles, repeating pharyngitis, sinusitis, otitis, and frequent acute respiratory infection.

Polyclonal IgGs from 10 healthy controls were inactive in DNA hydrolysis (Odintsova et al., 2006a). Similar results were obtained earlier for several groups of 10-20 healthy humans used as controls in the studies of DNase Abs from the sera of patients with AI diseases (Nevinsky *et al.,* 2003, 2005, 2010a, 2010b and refs therein).The type of plasmid supercoiled (sc) DNA hydrolysis by AIDS pIgGs did not depend on the Ab concentration and the rate of the hydrolysis linearly increased with the increase in IgG concentration and time of incubation (Odintsova et al., 2006a). Fig. 5 illustrates a cleavage of plasmid DNA (14 g/ml) by Abs (0.3 mg/ml) from several AIDS patients after 4 h of incubation. One can see that in this period some Abs cause only single breaks in one strand of supercoiled DNA (lanes 1-3), whereas others cause multiple breaks and as a result the formation of linear DNA (lanes 4-6). The most active Abs hydrolyze DNA into short and medium length ODNs (lanes 7-10). It should be mentioned that Fig. 5 in principle illustrates a range of possible changes of the relative DNase activities for patients with not only AIDS but also with different AI diseases and viral pathologies previously analyzed (Nevinsky *et al.,* 2000a, 2003, 2005, 2010a, 2010b and refs therein). When passing from one pathology to another only the values of a relative percent of patients with low, middle and high DNase activities is usually changed.

Fig. 5. DNase activities of catalytic AIDS IgGs from different patients in the cleavage of supercoiled (sc) and nicked (n) plasmid DNA (14 g/ml). Lanes 1–10, IgGs (0.3 mg/ml; incubation for 4 h) from the sera of 10 different patients; C1, scDNA incubated alone; C2 and C3, scDNA incubated with Ab from the sera of two healthy donors.

The efficiency of DNA cleavage was calculated from the increase of DNA in the band of scDNA (10-40 % of initial DNA hydrolysis); the measured RAs (%) for IgGs were normalized to standard conditions (0.1 mg/ml Abs, 2 h) and a complete transition of scDNA to its relaxed form was taken for 100%. While four AIDS IgG preparations were completely inactive, the remaining 106 IgG samples (96 %) demonstrated the RAs from 5 to 100% of scDNA hydrolysis (Table 1).

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 163

two subgroups with either rapid or slow progression of the disease. According to the recommendations of the Center of Disease Control and Prevention, the transition time from GL to pre-AIDS stage (≤ 2 and > 2 years, respectively) was used as the measure of the disease rate of progression. The number of GL patients demonstrating a detectable proteolytic activity was comparable in the case of rapid (41.7 %) and slow (47.8%) progression, while DNase activity was observed in 41.3 % of the patients with rapid progression and only in 29.5% of the patients with slow progression. In the pre-AIDS group, the number of IgGs with DNase and proteolytic activity was slightly higher in patients with rapid progression (55.8 and 58.1 %, respectively) than in patients with slow (50.7 and 51.8 %)

On first glance, high activity of IgGs from the blood of AIDS patients in hydrolysis of βcasein (which is not a typical component of human blood) is unexpected. However, it was recently shown by 2D electrophoresis that six of nine sera from AIDS patients contained Abs against casein, and five against human milk lactalbumin (Goldfarb, 2001). Thereby, the activation of β-casein synthesis in AIDS patients driven by not yet understood factor can not be excluded. It is interesting that mRNA corresponding to the gene encoding for β-casein is produced in mouse T-killer cells (also for unknown reason) (Grusby et al., 1990). In this way, it can not be excluded that genes, encoding for β-casein, as well as this protein itself, can play a special (but not yet known) role in the HIV virus life cycle, its replication, or

At the same time, so far Abzs hydrolyzing HSA were found only in AIDS patients. A possible reason of production of HSA-hydrolyzing Abs in AIDS patients is also not known. Later the RAs of AIDS IgGs (Baranova et al., 2009) and IgMs (Baranova et al., 2010) in the hydrolysis of IN were analyzed. Sera of 19 HIV-infected patients (18-40 yr old; men and women) including 13 at the stage of pre-AIDS and 6 at the stage of GL were used to study IN-hydrolyzing activity of IgGs, while 18 Ab preparations corresponding to pre-AIDS stage

pIgGs and pIgMs from ten control healthy donors were unable to catalyze IN hydrolysis. Interestingly, 11 of 13 IgGs from patients with pre-AIDS (84.6 %) and 6 of 6 (100 %) with GL demonstrated detectable of high IN-hydrolyzing activity (Baranova et al., 2009). There was no statistically significant difference in the IgG RAs between the two groups of patients; average values of IgG IN-hydrolyzing RAs were 1.99 ± 1.68 for pre-AIDS and 3.4 ± 1.31 (M IN/1h)/mg of Abs for GL patients. All 16 IgMs purified from patients with pre-AIDS (100%) and 6 of 8 IgMs (75%) from patients with GL demonstrated high or detectable INhydrolyzing activity (Baranova et al., 2010). There was no statistically significant difference (*p* = 0.71) in the IgM RAs between the two groups of patients; the average values of IgM INhydrolyzing RAs were 3.8 ± 2.2 M IN per hour per mg of Abs (range 0.3–7.3 M IN per hour per mg of Abs) for pre-AIDS and 3.3 ± 2.6 M IN per hour per mg of Abs (range 0–8.1

Overall, in the case of Abs with DNase, casein-, and IN-hydrolyzing activity we have found only a negligible difference in the RAs of Abs from HIV-infected patients at the GL and pre-AIDS stages. However, it is not surprising and agrees with the published data that a detection of Abzs is the earliest indicator of the development of many AI diseases in humans and animals (Andryushkova et al., 2006, 2007, 2009; Nevinsky et al., 2005, 2010a, 2010b, and refs therein). According to our data, various catalytic activities of Abzs are usually very easily detectable at the onset of AI diseases when the total concentrations of non-catalytic

progression of the disease.

development of AI reactions in AIDS patients.

M IN per hour per mg of Abs) for GL.

and 8 preparations to GL were used to analyze RAs of IgMs.

On the fist step we have analyzed the RAs of proteolytic activity of AIDS pIgGs in the hydrolysis of many different proteins including HIV RT, HSA, and human -casein (Odintsova et al., 2006b). It was shown that among all proteins used, IgGs from different HIV-infected patients with detectable or high rate hydrolyze only HIV-1 reverse transcriptase (RT), human serum albumin (HSA), and human -casein. Interestingly, the highest rate of the hydrolysis was observed for -casein (Odintsova et al., 2006b). Therefore, the RA of 110 AIDS IgGs in the hydrolysis of casein was analyzed. The efficiency of casein cleavage was analysed by SDS-PAGE and calculated from the decrease in the band of nonhydrolyzed casein taking into account the control reaction incubated in the absence of IgGs.

\*Percent of total number of patients (110) is given in parenthesis.

\*\*A complete transition of 14 g/ml scDNA to its relaxed form (0.1 mg/ml IgGs, 2 h) was taken for 100%. \*\*\*A complete hydrolysis of 0.1 mg/ml human -casein (0.1 mg/ml IgGs, 2 h) was taken for 100%.

Table 1. The relative catalytic activities of AIDS IgGs in the hydrolysis of scDNA and casein in the case of total group and patients with different stages of diseases development.

Purified pIgGs from ten healthy donors were unable to catalyze casein hydrolysis, whereas 105 of 110 IgGs (95 %) demonstrated high or detectable casein-hydrolyzing activity (Table 1). With the development of the disease at the stage of pre-AIDS, IgGs from some patients demonstrated a high DNase activity (≥80 % of DNA hydrolysis) but the average RA was 44.7 % 21.3 % (Table 1). At the stage of generalized lymphadenopathy, IgGs from 4 of 45 patients (8.8 %) did not possess detectable DNase activity. However, the average RA value of DNase activity for this group of patients (40.2 % 26.3 %) was comparable with that for the pre-AIDS group. At the same time, the number of IgG preparations with very high activities (≥80 % of DNA hydrolysis) was significantly lower in the GL group. At the GL stage, 5 of 45 (11 %) Abs were completely inactive, while several IgGs demonstrated high RA in the hydrolysis of casein (up to 86.7%); the average RA for all 45 patients was 53.6 % 22.6 % (Table 1). All 65 IgGs from patients with pre-AIDS were catalytically active, but the average RA (51.5 % 25.9 %) was comparable with that for IgGs from patients with GL (Table 1). Each group of patients corresponding to GL and pre-AIDS stage was divided into

On the fist step we have analyzed the RAs of proteolytic activity of AIDS pIgGs in the hydrolysis of many different proteins including HIV RT, HSA, and human -casein (Odintsova et al., 2006b). It was shown that among all proteins used, IgGs from different HIV-infected patients with detectable or high rate hydrolyze only HIV-1 reverse transcriptase (RT), human serum albumin (HSA), and human -casein. Interestingly, the highest rate of the hydrolysis was observed for -casein (Odintsova et al., 2006b). Therefore, the RA of 110 AIDS IgGs in the hydrolysis of casein was analyzed. The efficiency of casein cleavage was analysed by SDS-PAGE and calculated from the decrease in the band of nonhydrolyzed casein taking into account the control reaction incubated in the absence of IgGs.

> Number of IgG preparations with DNase activity\*\*

lymphadenopathy (45) 40.2 % ± 26.3 % 53.6 % 22.6 % pre-AIDS (65) 44.7 % ± 21.3 % 51.5 % 25.9 %

\*\*A complete transition of 14 g/ml scDNA to its relaxed form (0.1 mg/ml IgGs, 2 h) was taken for 100%. \*\*\*A complete hydrolysis of 0.1 mg/ml human -casein (0.1 mg/ml IgGs, 2 h) was taken for 100%. Table 1. The relative catalytic activities of AIDS IgGs in the hydrolysis of scDNA and casein in the case of total group and patients with different stages of diseases development. Purified pIgGs from ten healthy donors were unable to catalyze casein hydrolysis, whereas 105 of 110 IgGs (95 %) demonstrated high or detectable casein-hydrolyzing activity (Table 1). With the development of the disease at the stage of pre-AIDS, IgGs from some patients demonstrated a high DNase activity (≥80 % of DNA hydrolysis) but the average RA was 44.7 % 21.3 % (Table 1). At the stage of generalized lymphadenopathy, IgGs from 4 of 45 patients (8.8 %) did not possess detectable DNase activity. However, the average RA value of DNase activity for this group of patients (40.2 % 26.3 %) was comparable with that for the pre-AIDS group. At the same time, the number of IgG preparations with very high activities (≥80 % of DNA hydrolysis) was significantly lower in the GL group. At the GL stage, 5 of 45 (11 %) Abs were completely inactive, while several IgGs demonstrated high RA in the hydrolysis of casein (up to 86.7%); the average RA for all 45 patients was 53.6 % 22.6 % (Table 1). All 65 IgGs from patients with pre-AIDS were catalytically active, but the average RA (51.5 % 25.9 %) was comparable with that for IgGs from patients with GL (Table 1). Each group of patients corresponding to GL and pre-AIDS stage was divided into

\*Percent of total number of patients (110) is given in parenthesis.

0 4 (4 %)\* 5 (5 %) 5 – 20 21 (19 %) 11 (10 %) 21 – 40 33 (30 %) 24 (21.4 %) 41 – 60 18 (16.4 %) 25 (22.7 %) 61 – 80 7 (6.4 %) 20 (18.2 %) 81 – 100 17 (15.5 %) 25 (22.7 %)

Average DNase RA Average protease RA

Number of IgG preparations with casein-hydrolyzing

activity\*\*\*

Relative activity, % of scDNA hydrolysis

Number of preparations and

stage of disease

Generalized

two subgroups with either rapid or slow progression of the disease. According to the recommendations of the Center of Disease Control and Prevention, the transition time from GL to pre-AIDS stage (≤ 2 and > 2 years, respectively) was used as the measure of the disease rate of progression. The number of GL patients demonstrating a detectable proteolytic activity was comparable in the case of rapid (41.7 %) and slow (47.8%) progression, while DNase activity was observed in 41.3 % of the patients with rapid progression and only in 29.5% of the patients with slow progression. In the pre-AIDS group, the number of IgGs with DNase and proteolytic activity was slightly higher in patients with rapid progression (55.8 and 58.1 %, respectively) than in patients with slow (50.7 and 51.8 %) progression of the disease.

On first glance, high activity of IgGs from the blood of AIDS patients in hydrolysis of βcasein (which is not a typical component of human blood) is unexpected. However, it was recently shown by 2D electrophoresis that six of nine sera from AIDS patients contained Abs against casein, and five against human milk lactalbumin (Goldfarb, 2001). Thereby, the activation of β-casein synthesis in AIDS patients driven by not yet understood factor can not be excluded. It is interesting that mRNA corresponding to the gene encoding for β-casein is produced in mouse T-killer cells (also for unknown reason) (Grusby et al., 1990). In this way, it can not be excluded that genes, encoding for β-casein, as well as this protein itself, can play a special (but not yet known) role in the HIV virus life cycle, its replication, or development of AI reactions in AIDS patients.

At the same time, so far Abzs hydrolyzing HSA were found only in AIDS patients. A possible reason of production of HSA-hydrolyzing Abs in AIDS patients is also not known.

Later the RAs of AIDS IgGs (Baranova et al., 2009) and IgMs (Baranova et al., 2010) in the hydrolysis of IN were analyzed. Sera of 19 HIV-infected patients (18-40 yr old; men and women) including 13 at the stage of pre-AIDS and 6 at the stage of GL were used to study IN-hydrolyzing activity of IgGs, while 18 Ab preparations corresponding to pre-AIDS stage and 8 preparations to GL were used to analyze RAs of IgMs.

pIgGs and pIgMs from ten control healthy donors were unable to catalyze IN hydrolysis. Interestingly, 11 of 13 IgGs from patients with pre-AIDS (84.6 %) and 6 of 6 (100 %) with GL demonstrated detectable of high IN-hydrolyzing activity (Baranova et al., 2009). There was no statistically significant difference in the IgG RAs between the two groups of patients; average values of IgG IN-hydrolyzing RAs were 1.99 ± 1.68 for pre-AIDS and 3.4 ± 1.31 (M IN/1h)/mg of Abs for GL patients. All 16 IgMs purified from patients with pre-AIDS (100%) and 6 of 8 IgMs (75%) from patients with GL demonstrated high or detectable INhydrolyzing activity (Baranova et al., 2010). There was no statistically significant difference (*p* = 0.71) in the IgM RAs between the two groups of patients; the average values of IgM INhydrolyzing RAs were 3.8 ± 2.2 M IN per hour per mg of Abs (range 0.3–7.3 M IN per hour per mg of Abs) for pre-AIDS and 3.3 ± 2.6 M IN per hour per mg of Abs (range 0–8.1 M IN per hour per mg of Abs) for GL.

Overall, in the case of Abs with DNase, casein-, and IN-hydrolyzing activity we have found only a negligible difference in the RAs of Abs from HIV-infected patients at the GL and pre-AIDS stages. However, it is not surprising and agrees with the published data that a detection of Abzs is the earliest indicator of the development of many AI diseases in humans and animals (Andryushkova et al., 2006, 2007, 2009; Nevinsky et al., 2005, 2010a, 2010b, and refs therein). According to our data, various catalytic activities of Abzs are usually very easily detectable at the onset of AI diseases when the total concentrations of non-catalytic

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 165

(Andryushkova et al., 2006, 2007, 2009). Immunization of healthy mice with DNA also leads to production of DNase Abzs; however, it is associated only with increased lymphocyte proliferation and suppression of apoptosis of lymphocytes in different organs (especially spleen), but not with a change in differentiation of the bone marrow cells. Immune processes after immunization of mammals with bacterial DNA, proteins, polysaccharides during many infectious diseases may be considered similar to those after immunization of healthy mice with DNA, different proteins and enzymes. According to theoretical analysis, the adaptive improvement of the catalytic turnover is limited by the rate of B cell receptor signal transduction, as rapid release of antigen fragments from catalytic B cell receptors aborts clonal selection, but production of catalysts can occur at increased levels under conditions of rapid B cell signaling in AI disease (Paul et al., 2006). In addition, the RAs of DNase Abs increased with the progress of the AI pathology, while the time course of immunization associated with some infections is usually not so long as compared with AI diseases, which can have chronic character. In contrast to AI diseases, treatment and recovery of patients with bacterial infections usually eliminates Abzs with various activities. In addition, Abzs may have protective functions in patients with bacterial infections. It was shown that the presence of IgG endowed with serine protease-like activity in the plasma of patients with

sepsis strongly correlates with their survival (Lacroix-Desmazes et al., 2005).

these bacterial and viral components.

**5. Extreme diversity of AIDS abzymes 5.1 Structural diversity of AIDS abzymes**

AI processes.

In contrast to DNase abzymes, the polysaccharide-hydrolyzing Abs are usually present even in the sera of healthy humans and their activity remarkably increases in the sera of patients and animals with different AI diseases (Andryushkova et al., 2006; 2007) and especially with pathologies caused by viral infections (Buneva V.N., personal communication). Formation of specific Abs against DNA and other components of bacteria and some viruses during infections of healthy mammals suggests that the specific catalytic Abs can mostly hydrolyze

It is possible that co-action of Abzs with proteolytic and polysaccharide-hydrolyzing activities can at least partially degrade bacterial cell walls and viral particles and facilitate the entry of Abzs into the bacterial cells and hydrolysis of bacterial DNA, proteins and other components. This cooperative action of abzymes with different catalytic activities may have a protective effect against diseases caused by bacterial and viral infections. At the same time, in contrast to many viral and bacterial infections, HIV-infection stimulates AI reactions. Therefore, at the first stage of AIDS development catalytic Abzs against different viral components can protect humans, similarly to the situation in bacterial and viral infections that do not stimulate AI reactions. Later, due to molecular mimicry between viral and host antigens, viral antigens can affect hematopoietic stem cells and trigger the development of

DNase, RNase, ATPase, amylase, and protease Abzs may show very different contributions of variable domains of H- and L-chains to their active centers. Chromatographically separated light chains of IgGs from the sera of asthma patients were found to be active in the hydrolysis of VIP (Sun et al., 1994; 1997). The light chain of the VIP Abz was expressed in bacteria, purified, and found to possess an intrinsic catalytic activity (Tyutyulkova et al., 1996). The Abz-dependent hydrolysis of DNA and RNA by isolated light chains of IgGs from SLE, MS, asthma, and other AI patients, as well as from MRL-lpr/lpr mice, is more

Abs to specific auto-antigens have not yet increased significantly and correspond to their ranges for healthy donors. At the early stages of AI diseases, the repertoire of Abzs is usually relatively small but it greatly increases with the progress of the disease, leading to the generation of catalytically diverse abzymes with different activities and functions (Nevinsky *et al.,* 2003, 2005, 2010a, 2010b and refs therein). In addition, AI reactions in the case of some viral diseases including AIDS patients are in some extent similar to AI diseases. At the same time, immunization of AI mice produces an unexpectedly high increase in the number of clones secreting various auto-Abs, including Abzs, in comparison with normal mice (Nishi, 2002; Tawfik et al., 2002).

HIV-1 RT- and IN-hydrolzying pIgGs and IgMs from HIV-infected patients were the first examples of catalytic Abzs produced in humans against viral proteins after a viral infection (Odintsova et al., 2006b; Baranova et al., 2009, 2010). In addition, it was shown for the first time that HIV infection stimulates autoimmune reactions leading to the formation of Abzs that hydrolyze at least two human proteins, HSA and casein. It is known that HIV infection stimulates the development of many AI diseases (Zandman-Goddard et al., 2002). One can suppose that in some other viral and bacterial infections may induce similar processes to some extent.

During many infections, the human organism is exposed to different bacterial components including protein, DNA, RNA, and polysaccharides (Ternynck et al., 1991; Unterkircher et al., 1993; Matsiota-Bernard et al., 1993, 1996; Hentati et al., 1994; Boekel et al., 1997; Wun et al., 2001). Because of their ability to bind a variety of exogenous antigens, including bacterial and viral ones, natural Abs play a major role in the primary line of defense against infections. Some results suggest that the synthesis of auto-Abs and Abs directed against bacterial antigens at least partially follow distinct pathways, but with the existing experimental data it is impossible to determine unambiguously whether these two Ab populations are produced by the same or distinct B-cell subpopulations (Matsiota-Bernard et al., 1993). Recently, DNase activity in the patients with diseases caused by several bacterial infections has been analyzed (Parkhomenko et al., 2008). The catalytic activities were significantly lower than in patients with different AI pathologies and increased in the following order: streptococcal infection (erysipelas) < urogenital chlamydiosis associated with arthritis (Reiter's disease) < meningococcal meningitis < shigellosis < suppurative surgical infections caused by *Staphylococcus aureus* < suppurative surgical infections caused by epidermal staphylococci < urogenital ureaplasmosis associated with reactive arthritis.

In addition, DNA-hydrolyzing IgGs was found in the sera of patients with hepatitis (Baranovskii et al., 1997) and tick bone encephalitis (Parkhomenko et al., 2010-11). Interestingly, TBE like HIV infection of humans stimulate formation of Abzs with several proteolytic activities (Parkhomenko T., personal communication). The RAs of IgGs in the hydrolysis of DNA increased in the following order: diabetes ≤ bacterial infections ≤ viral hepatitis < polyarthritis < Hashimoto's thyroiditis < AIDS ≤ MS < SLE (Nevinsky *et al.,* 2003, 2005, 2010a, 2010b and refs therein).

Taking these observations together, we suggest that the specific activity of polyclonal Abs from the sera of patients with diseases caused by bacterial infections are usually lower than those for typical AI diseases and most probably they can differ in their biological functions. It was shown that the specific reorganization of immune system during the spontaneous development of a profound SLE-like pathology in MRL-lpr/lpr mice is associated with changes in the differentiation profile and the level of proliferation of bone marrow hematopoietic stem cells and with production of DNase, ATPase, and amylase Abzs

Abs to specific auto-antigens have not yet increased significantly and correspond to their ranges for healthy donors. At the early stages of AI diseases, the repertoire of Abzs is usually relatively small but it greatly increases with the progress of the disease, leading to the generation of catalytically diverse abzymes with different activities and functions (Nevinsky *et al.,* 2003, 2005, 2010a, 2010b and refs therein). In addition, AI reactions in the case of some viral diseases including AIDS patients are in some extent similar to AI diseases. At the same time, immunization of AI mice produces an unexpectedly high increase in the number of clones secreting various auto-Abs, including Abzs, in comparison with normal

HIV-1 RT- and IN-hydrolzying pIgGs and IgMs from HIV-infected patients were the first examples of catalytic Abzs produced in humans against viral proteins after a viral infection (Odintsova et al., 2006b; Baranova et al., 2009, 2010). In addition, it was shown for the first time that HIV infection stimulates autoimmune reactions leading to the formation of Abzs that hydrolyze at least two human proteins, HSA and casein. It is known that HIV infection stimulates the development of many AI diseases (Zandman-Goddard et al., 2002). One can suppose that in some other viral and bacterial infections may induce similar processes to

During many infections, the human organism is exposed to different bacterial components including protein, DNA, RNA, and polysaccharides (Ternynck et al., 1991; Unterkircher et al., 1993; Matsiota-Bernard et al., 1993, 1996; Hentati et al., 1994; Boekel et al., 1997; Wun et al., 2001). Because of their ability to bind a variety of exogenous antigens, including bacterial and viral ones, natural Abs play a major role in the primary line of defense against infections. Some results suggest that the synthesis of auto-Abs and Abs directed against bacterial antigens at least partially follow distinct pathways, but with the existing experimental data it is impossible to determine unambiguously whether these two Ab populations are produced by the same or distinct B-cell subpopulations (Matsiota-Bernard et al., 1993). Recently, DNase activity in the patients with diseases caused by several bacterial infections has been analyzed (Parkhomenko et al., 2008). The catalytic activities were significantly lower than in patients with different AI pathologies and increased in the following order: streptococcal infection (erysipelas) < urogenital chlamydiosis associated with arthritis (Reiter's disease) < meningococcal meningitis < shigellosis < suppurative surgical infections caused by *Staphylococcus aureus* < suppurative surgical infections caused by epidermal staphylococci < urogenital ureaplasmosis associated with reactive arthritis. In addition, DNA-hydrolyzing IgGs was found in the sera of patients with hepatitis (Baranovskii et al., 1997) and tick bone encephalitis (Parkhomenko et al., 2010-11). Interestingly, TBE like HIV infection of humans stimulate formation of Abzs with several proteolytic activities (Parkhomenko T., personal communication). The RAs of IgGs in the hydrolysis of DNA increased in the following order: diabetes ≤ bacterial infections ≤ viral hepatitis < polyarthritis < Hashimoto's thyroiditis < AIDS ≤ MS < SLE (Nevinsky *et al.,* 2003,

Taking these observations together, we suggest that the specific activity of polyclonal Abs from the sera of patients with diseases caused by bacterial infections are usually lower than those for typical AI diseases and most probably they can differ in their biological functions. It was shown that the specific reorganization of immune system during the spontaneous development of a profound SLE-like pathology in MRL-lpr/lpr mice is associated with changes in the differentiation profile and the level of proliferation of bone marrow hematopoietic stem cells and with production of DNase, ATPase, and amylase Abzs

mice (Nishi, 2002; Tawfik et al., 2002).

2005, 2010a, 2010b and refs therein).

some extent.

(Andryushkova et al., 2006, 2007, 2009). Immunization of healthy mice with DNA also leads to production of DNase Abzs; however, it is associated only with increased lymphocyte proliferation and suppression of apoptosis of lymphocytes in different organs (especially spleen), but not with a change in differentiation of the bone marrow cells. Immune processes after immunization of mammals with bacterial DNA, proteins, polysaccharides during many infectious diseases may be considered similar to those after immunization of healthy mice with DNA, different proteins and enzymes. According to theoretical analysis, the adaptive improvement of the catalytic turnover is limited by the rate of B cell receptor signal transduction, as rapid release of antigen fragments from catalytic B cell receptors aborts clonal selection, but production of catalysts can occur at increased levels under conditions of rapid B cell signaling in AI disease (Paul et al., 2006). In addition, the RAs of DNase Abs increased with the progress of the AI pathology, while the time course of immunization associated with some infections is usually not so long as compared with AI diseases, which can have chronic character. In contrast to AI diseases, treatment and recovery of patients with bacterial infections usually eliminates Abzs with various activities. In addition, Abzs may have protective functions in patients with bacterial infections. It was shown that the presence of IgG endowed with serine protease-like activity in the plasma of patients with sepsis strongly correlates with their survival (Lacroix-Desmazes et al., 2005).

In contrast to DNase abzymes, the polysaccharide-hydrolyzing Abs are usually present even in the sera of healthy humans and their activity remarkably increases in the sera of patients and animals with different AI diseases (Andryushkova et al., 2006; 2007) and especially with pathologies caused by viral infections (Buneva V.N., personal communication). Formation of specific Abs against DNA and other components of bacteria and some viruses during infections of healthy mammals suggests that the specific catalytic Abs can mostly hydrolyze these bacterial and viral components.

It is possible that co-action of Abzs with proteolytic and polysaccharide-hydrolyzing activities can at least partially degrade bacterial cell walls and viral particles and facilitate the entry of Abzs into the bacterial cells and hydrolysis of bacterial DNA, proteins and other components. This cooperative action of abzymes with different catalytic activities may have a protective effect against diseases caused by bacterial and viral infections. At the same time, in contrast to many viral and bacterial infections, HIV-infection stimulates AI reactions. Therefore, at the first stage of AIDS development catalytic Abzs against different viral components can protect humans, similarly to the situation in bacterial and viral infections that do not stimulate AI reactions. Later, due to molecular mimicry between viral and host antigens, viral antigens can affect hematopoietic stem cells and trigger the development of AI processes.

## **5. Extreme diversity of AIDS abzymes**

#### **5.1 Structural diversity of AIDS abzymes**

DNase, RNase, ATPase, amylase, and protease Abzs may show very different contributions of variable domains of H- and L-chains to their active centers. Chromatographically separated light chains of IgGs from the sera of asthma patients were found to be active in the hydrolysis of VIP (Sun et al., 1994; 1997). The light chain of the VIP Abz was expressed in bacteria, purified, and found to possess an intrinsic catalytic activity (Tyutyulkova et al., 1996). The Abz-dependent hydrolysis of DNA and RNA by isolated light chains of IgGs from SLE, MS, asthma, and other AI patients, as well as from MRL-lpr/lpr mice, is more

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 167

iodoacetamide (a specific inhibitor of thiol proteases) usually has no remarkable effect on their proteolytic activity (Paul et al., 1989; Li et al., 1995; Kalaga et al., 1995; Odintsova et al., 2006; 2011; Polosukhina et al., 2004, 2005, 2006). Surprisingly, however, IN-hydrolyzing activity of all 10 IgGs was inhibited by iodoacetamide by 12-98 % (Baranova et al., 2009).

**1 2 3 4 5 6 7**

Fig. 6. SDS-PAGE analysis of a decrease in the intensity of protein band after its incubation with pIgGs from AIDS patients in different conditions. IN was incubated alone (1); in the presence of AIDS pIgGs from the sera of six different AIDS patients (IgG-12 – IgG19) and in

Similar surprising situation was observed for ten AIDS pIgMs with IN-hydrolyzing activity (Table 2) (Baranova et al., 2010). Leupeptin, significantly inhibited the proteolytic activity of only two of ten individual AIDS IgMs, and very weak inhibition was observed for one more preparation (Table 2). A specific inhibitor of acidic proteases, pepstatin A, significantly inhibited the IN-hydrolyzing activity of only three pIgMs and demonstrated weak inhibition of two preparations (Table 2). Surprisingly, a significant inhibition of serine protease-like activity by AEBSF was found only for two and weak suppression of the activity for another of ten AIDS IgMs. Proteolytic activity of five of ten AIDS IgMs was inhibited by 33–91% after incubating the IgMs with 0.01 M EDTA, while this chelating reagent at 0.1 M concentration decreased the relative activity of six preparations by 64–98% and inhibited three more preparations for ~8-10% (Table 2). As for AIDS IgGs, iodoacetamide inhibited the IN-hydrolyzing activity of all ten of ten Abs by 30–99% (Table 2). The inhibition of AIDS IgGs and IgMs with EDTA was comparable with that for IgGs from patients with MS

Iodoacetamide, a specific inhibitor of thiol proteases, usually does not significantly affect the activity of proteolytic Abzs (≤ 3–7% inhibition) (see above). Therefore, it was surprising that the IN-hydrolyzing activity of AIDS IgGs was suppressed by iodoacetamide in all 100% preparations by 12–98% (average value 65.7 ± 20.6%) in a stark contrast with other known proteolytic Abzs. A similar result was observed for AIDS IgMs; iodoacetamide suppressed the IN-hydrolyzing activity by 30–99% (average value 75.6 ± 21.2%) in all ten Ab preparations. Interestingly, there was no statistically significant difference in the inhibition of AIDS pIgGs and pIgMs by iodoacetamide (*p* = 0.2). Our findings support the idea that the pools of pIgGs and IgMs of AIDS patients can contain IN-hydrolyzing Abzs of four types

the absence of other components (2) or in the presence of AEBSF (3), leupeptin (4), iodoacetamide (5), EDTA (6), and pepstatin A (lane 7). Arrows indicate the positions of

IN

**IgG12**

**IgG13**

**IgG14**

**IgG15**

**IgG16**

**IgG19**

molecular mass markers.

 A similar situation was observed for human milk IgGs and sIgAs with DNase and RNase activities (Kanyshkova et al., 1997; Nevinsky et al., 2000a). In addition, both H and L-chains of sIgAs had affinity to DNA-cellulose but only L-subunits hydrolyzed DNA and RNA (Nevinsky et al., 2000c). At the same time, it was demonstrated that the catalytic center of recombinant variable fragment (scFv) of DNase IgGs from AI-prone MRL-lpr/lpr mice may be located at the interface between the light and heavy chains and that after separation both of these chains are able to hydrolyze DNA (Kim et al., 2006).

It was shown that only separated light chains of AIDS IgGs hydrolyze DNA, while heavy chains is catalytically inactive (Odintsova et al., 2006a). On the contrary, both light and heavy chains of mouse IgGs after separation were active in the ATP hydrolysis (Andryushkova et al., 2009). Intact rat pIgGs and their separated H- and L-chains possess both peroxidase and oxidoreductase catalytic activity (Nevinsky et al., 2010a). The observed IN-hydrolyzing activity of AIDS pIgM L- and H-chains separated by SDS-PAGE (Fig. 3) may have different underlying causes. First, it is possible that these pIgMs contain a mixture of Abzs with only light or only heavy chains being catalytically active. However, similarly to mouse monoclonal DNase (Kim et al., 2006), catalytic centers of AIDS IgMs hydrolyzing IN may be located at the interface between the light and heavy chains, with both separated chains capable of hydrolysis of IN.

From the crystal structure of a catalytic Ab with esterase-like activity, it was concluded that the ligand *p*-nitrophenyl acetate interacts with amino acid residues of both light and heavy chains of Abzs and that both types of subunits are required for catalysis (Golinelli-Pimpaneau et al., 1994). Taken together, it is obvious that light and heavy chains of different Abzs including AIDS Abs can contribute to the active sites of Abzs in different ways.

The next question concerning the structural diversity of AIDS Abzs relates to the type of the proteolytic activity of their catalytic sites. Proteolytic IgGs from the sera of patients with asthma hydrolyzing VIP (Paul et al., 1989), Hashimoto thyroiditis and rheumatoid arthritis hydrolyzing thyroglobulin (Li eta l., 1995; Kalaga et al., 1995) are serine-like proteases, and their activity is most strongly reduced after incubation with specific serine protease inhibitors PMSF or AEBSF. It was shown that casein-hydrolyzing sIgAs from human milk (Odintsova et al., 2006; 2011) and hMBP-hydrolyzing IgGs and IgMs from the sera of patients with MS (Polosukhina et al., 2004, 2005, 2006) contain not only Ab subfractions with serine-like, but also specific subfractions with metal-dependent activity.

We have analyzed the type of proteolytic activity of AIDS pIgGs and pIgMs in the hydrolysis of IN and -casein. It was shown, that in contrast to milk sIgAs (Odintsova et al., 2006; 2011) and similarly to several other proteolytic Abzs, only specific inhibitor of serine protease AEBSF significantly suppress AIDS pIgG-dependent hydrolysis of -casein, while EDTA has no significant effect.

We have analyzed the type of IN-hydrolyzing activity of ten AIDS pIgGs; several typical examples are given in Fig. 6 (Baranova et al., 2009). Leupeptin, an inhibitor of many different proteases, demonstrated significant inhibition of proteolytic activity of only 2 of 10 individual AIDS IgGs (Table 2). A similar situation was observed for a specific inhibitor of acidic proteases, pepstatin A, which significantly inhibited IN-hydrolyzing activity of only two IgGs. Surprisingly, a significant inhibition of serine protease-like activity was also found only for 2 of 10 AIDS IgGs (Table 2). Proteolytic activity of five of ten AIDS IgGs was inhibited by 40-96 % after incubating the IgGs with EDTA. An incubation of IgGs with

efficient than by intact Abs (Dubrovskaya et al., 2003; Galvita et al., 2007; Andrievskaya et al., 2000, 2002; Baranovskii et al., 2001; Nevinsky et al., 2005, 2010a, 2010b). A similar situation was observed for human milk IgGs and sIgAs with DNase and RNase activities (Kanyshkova et al., 1997; Nevinsky et al., 2000a). In addition, both H and L-chains of sIgAs had affinity to DNA-cellulose but only L-subunits hydrolyzed DNA and RNA (Nevinsky et al., 2000c). At the same time, it was demonstrated that the catalytic center of recombinant variable fragment (scFv) of DNase IgGs from AI-prone MRL-lpr/lpr mice may be located at the interface between the light and heavy chains and that after separation both of these

It was shown that only separated light chains of AIDS IgGs hydrolyze DNA, while heavy chains is catalytically inactive (Odintsova et al., 2006a). On the contrary, both light and heavy chains of mouse IgGs after separation were active in the ATP hydrolysis (Andryushkova et al., 2009). Intact rat pIgGs and their separated H- and L-chains possess both peroxidase and oxidoreductase catalytic activity (Nevinsky et al., 2010a). The observed IN-hydrolyzing activity of AIDS pIgM L- and H-chains separated by SDS-PAGE (Fig. 3) may have different underlying causes. First, it is possible that these pIgMs contain a mixture of Abzs with only light or only heavy chains being catalytically active. However, similarly to mouse monoclonal DNase (Kim et al., 2006), catalytic centers of AIDS IgMs hydrolyzing IN may be located at the interface between the light and heavy chains, with both separated

From the crystal structure of a catalytic Ab with esterase-like activity, it was concluded that the ligand *p*-nitrophenyl acetate interacts with amino acid residues of both light and heavy chains of Abzs and that both types of subunits are required for catalysis (Golinelli-Pimpaneau et al., 1994). Taken together, it is obvious that light and heavy chains of different

The next question concerning the structural diversity of AIDS Abzs relates to the type of the proteolytic activity of their catalytic sites. Proteolytic IgGs from the sera of patients with asthma hydrolyzing VIP (Paul et al., 1989), Hashimoto thyroiditis and rheumatoid arthritis hydrolyzing thyroglobulin (Li eta l., 1995; Kalaga et al., 1995) are serine-like proteases, and their activity is most strongly reduced after incubation with specific serine protease inhibitors PMSF or AEBSF. It was shown that casein-hydrolyzing sIgAs from human milk (Odintsova et al., 2006; 2011) and hMBP-hydrolyzing IgGs and IgMs from the sera of patients with MS (Polosukhina et al., 2004, 2005, 2006) contain not only Ab subfractions with

We have analyzed the type of proteolytic activity of AIDS pIgGs and pIgMs in the hydrolysis of IN and -casein. It was shown, that in contrast to milk sIgAs (Odintsova et al., 2006; 2011) and similarly to several other proteolytic Abzs, only specific inhibitor of serine protease AEBSF significantly suppress AIDS pIgG-dependent hydrolysis of -casein, while

We have analyzed the type of IN-hydrolyzing activity of ten AIDS pIgGs; several typical examples are given in Fig. 6 (Baranova et al., 2009). Leupeptin, an inhibitor of many different proteases, demonstrated significant inhibition of proteolytic activity of only 2 of 10 individual AIDS IgGs (Table 2). A similar situation was observed for a specific inhibitor of acidic proteases, pepstatin A, which significantly inhibited IN-hydrolyzing activity of only two IgGs. Surprisingly, a significant inhibition of serine protease-like activity was also found only for 2 of 10 AIDS IgGs (Table 2). Proteolytic activity of five of ten AIDS IgGs was inhibited by 40-96 % after incubating the IgGs with EDTA. An incubation of IgGs with

Abzs including AIDS Abs can contribute to the active sites of Abzs in different ways.

serine-like, but also specific subfractions with metal-dependent activity.

chains are able to hydrolyze DNA (Kim et al., 2006).

chains capable of hydrolysis of IN.

 Surprisingly, however, IN-hydrolyzing activity of all 10 IgGs was inhibited by iodoacetamide by 12-98 % (Baranova et al., 2009).

Fig. 6. SDS-PAGE analysis of a decrease in the intensity of protein band after its incubation with pIgGs from AIDS patients in different conditions. IN was incubated alone (1); in the presence of AIDS pIgGs from the sera of six different AIDS patients (IgG-12 – IgG19) and in the absence of other components (2) or in the presence of AEBSF (3), leupeptin (4), iodoacetamide (5), EDTA (6), and pepstatin A (lane 7). Arrows indicate the positions of molecular mass markers.

Similar surprising situation was observed for ten AIDS pIgMs with IN-hydrolyzing activity (Table 2) (Baranova et al., 2010). Leupeptin, significantly inhibited the proteolytic activity of only two of ten individual AIDS IgMs, and very weak inhibition was observed for one more preparation (Table 2). A specific inhibitor of acidic proteases, pepstatin A, significantly inhibited the IN-hydrolyzing activity of only three pIgMs and demonstrated weak inhibition of two preparations (Table 2). Surprisingly, a significant inhibition of serine protease-like activity by AEBSF was found only for two and weak suppression of the activity for another of ten AIDS IgMs. Proteolytic activity of five of ten AIDS IgMs was inhibited by 33–91% after incubating the IgMs with 0.01 M EDTA, while this chelating reagent at 0.1 M concentration decreased the relative activity of six preparations by 64–98% and inhibited three more preparations for ~8-10% (Table 2). As for AIDS IgGs, iodoacetamide inhibited the IN-hydrolyzing activity of all ten of ten Abs by 30–99% (Table 2). The inhibition of AIDS IgGs and IgMs with EDTA was comparable with that for IgGs from patients with MS (Polosukhina et al., 2004, 2005, 2006).

Iodoacetamide, a specific inhibitor of thiol proteases, usually does not significantly affect the activity of proteolytic Abzs (≤ 3–7% inhibition) (see above). Therefore, it was surprising that the IN-hydrolyzing activity of AIDS IgGs was suppressed by iodoacetamide in all 100% preparations by 12–98% (average value 65.7 ± 20.6%) in a stark contrast with other known proteolytic Abzs. A similar result was observed for AIDS IgMs; iodoacetamide suppressed the IN-hydrolyzing activity by 30–99% (average value 75.6 ± 21.2%) in all ten Ab preparations. Interestingly, there was no statistically significant difference in the inhibition of AIDS pIgGs and pIgMs by iodoacetamide (*p* = 0.2). Our findings support the idea that the pools of pIgGs and IgMs of AIDS patients can contain IN-hydrolyzing Abzs of four types

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 169

leupeptin and AEBSF for IgM7, and pepstatin A and AEBSF for IgM13 (Table 2). Very

In principle it is possible that the pools of IgGs and IgMs from AIDS patients may be "cocktails" of Abz molecules, with each molecule possessing only one of four alternative types of proteolytic activity: serine-, acidic-, thiol-, or metal-dependent. Yet the effects of two, three, and four inhibitors of different protease types did not always add to 100% inhibition. Only in three of ten IgM preparations (IgM8, IgM9, and IgM11) this sum was less or comparable with 100% (76–114%), while for other seven IgMs it varied from 125% to 289% (Table 2). Three IgG preparations (IgG9, IgG10, and IgG13) demonstrated this sum from 249 to 255 % (Table 2). Since IgM9 had only thiol protease-like activity, and IgM8 and IgM11 could be significantly suppressed (94–99%) only with iodoacetamide but lost their activity only marginally in the presence of EDTA or pepstatin A (by 7–8 %), it is most likely that in these patients most of the Abz molecules possess only the thiol protease type of proteolytic activity (Table 2). However, since the proteolytic activity in seven of ten IgMs and five of ten IgGs was summarily suppressed by specific inhibitors of serine, acidic, metaldependent, and thiol proteases by more than 100% (110–289%, Table 2), it is possible that the immune system of HIV-infected patients produces anti-IN Abzs with a combined structure of the active center, carrying amino acid residues typical of different proteases. For example, we suggest that the pools of IgM4, IgM7, and IgM13 (209–289% of the summarized inhibition) contain IgM molecules with extremely complicated active centers containing structural elements of thiol and metal-dependent proteases, which may be additionally combined with structural elements of the active centers of acidic proteases (IgM4), serine proteases (IgM7), or both (IgM13). Similar suggestion is reason in the case of several AIDS

Theoretically, a mammalian immune system can produce up to 106 variants of Abs against one antigen. An extreme diversity of RNase and DNase IgG and/or IgM Abzs from the sera of patients with MS and SLE and autoimmune prone MRL-lpr/lpr mice was observed (Baranovskii *et al.,* 1998, 2001; Andrievskaya *et al*., 2000, 2002; Kuznetsova *et al.,* 2007; Nevinsky *et al.*, 2003, 2005, 2010a, 2010b). It was shown that different patients (and animals) may have a relatively small or an extremely large pool of polyclonal nuclease Abzs containing different relative amounts of light chains of - and -types, demonstrating maximal activity at various optimal pHs, having a different net charge, activated or not by different metal ions, characterized by different substrate specificities. MS IgGs of all four subclasses (IgG1-IgG4) were catalytically active in the hydrolysis of hMBP (Legostaeva *et al*.,

We have analyzed the pH dependencies of the initial rates of DNA hydrolysis by AIDS catalytic IgGs (Odintsova et al., 2006a). Fig. 7A demonstrates three pH dependencies of different types which were revealed for catalytic pIgGs from the sera of 3 different patients. In contrast to all human DNases having one pronounced pH optimum in hydrolysis of DNA (Baranovskii et al., 2004), catalytic Abs usually show high DNase activity at a wide range of pH values between 5.5-9.0. Nevertheless, as one can see from Fig. 7A, one of the IgG preparations has a pronounced optimum at pH 8.0; second at pH 7.5, while third one

comparable situations were observed for AIDS IgGs and IgMs (Table 2).

IgGs (Table 2) (Baranova et al., 2009, 2010).

**5.2 pH optima diversity of AIDS abzymes**

2010) and DNA (Parkhomenko *et al*., 2010).

demonstrates two marked pH optima at pH 7.5 and 8.5.

\*\*The decrease in the intensity of initial IN band estimated from SDS-PAGE electrophoresis data in the absence of inhibitor was taken for 100 %, for each preparation, a mean of 3 repeats are used. \*\*Sum of the effects of different compounds on the proteolytic activity

(leupeptin+Pepstatin+iodoacetamide + EDTA).

Table 2. Inhibition of proteolytic activity of individual IgGs and IgMs (from 10 AIDS patients) in the hydrolysis of HIV integrase by specific inhibitors of proteases of different types

resembling thiol, serine, acidic, and metal-dependent proteases, the ratio of which may be individual for every HIV-infected patient.

Interestingly, only IgM9 and three of IgGs (IgG11, IgG14, and IgG19) demonstrated significant inhibition by one inhibitor (iodoacetamide). At the same time other IgG and IgM preparations were sensitive to two or three inhibitors (Table 2). For example, IgM10 and IgM11 was strongly suppressed by iodoacetamide and EDTA; IgM23 was sensitive to AEBSF and iodoacetamide; IgM4 and IgM8 showed strong or at least some inhibition of the activity by three inhibitors (iodoacetamide, EDTA, and pepstatin A), while IgM12 was sensitive to leupeptin instead of pepstatin A (Table 2). Surprisingly, three of ten preparations (IgM5, IgM7, and IgM13) could be significantly inhibited by four different inhibitors. Of these, iodoacetamide and EDTA were common inhibitors for all three IgM preparations, while two other inhibitors were different: pepstatin A and leupeptin for IgM5,

AEBSF Leupeptin Pepstatin A Iodoacetamide 0.1 M

IgG9 42±5 74±8 51±5 85±9 0 252 IgG10 0 70±7 0 83±9 96±9 249 IgG11 0 0 0 66±7 0 66 IgG12 0 0 0 66±5 44±5 110 IgG13 0 0 59±6 98±8 98±8 255 IgG14 0 0 0 87±7 3±1 90 IgG15 0 11±3 0 33±4 45±4 89 IgG16 0 0 0 78±8 40±3 118 IgG17 49±5 0 0 12±1.5 0 61 IgG19 0 0 0 49±5 2±1 51 IgM4 0 0 48 ± 5 97 ± 8 98 ± 2 243 IgM5 0 47 ± 5 36 ± 5 36 ± 5 80 ± 7 199 IgM7 8 ± 1 68 ± 7 0 95 ± 9 94 ± 8 265 IgM8 0 0 7 ± 2 99 ± 8 8 ± 2 114 IgM9 0 0 0 76 ± 6 0 76 IgM10 0 0 0 83 ± 7 93 ± 8 176 IgM11 0 0 0 94 ± 7 8 ± 3 102 IgM12 0 7 ± 1 0 55 ± 5 98 ± 8 153 IgM13 46 ± 5 0 88 ± 9 91 ± 10 64 ± 4 289 IgM23 80 ± 8 0 5 ± 2 30± 5 10 ± 2 125 \*\*The decrease in the intensity of initial IN band estimated from SDS-PAGE electrophoresis data in the

absence of inhibitor was taken for 100 %, for each preparation, a mean of 3 repeats are used.

in the hydrolysis of HIV integrase by specific inhibitors of proteases of different types

Table 2. Inhibition of proteolytic activity of individual IgGs and IgMs (from 10 AIDS patients)

resembling thiol, serine, acidic, and metal-dependent proteases, the ratio of which may be

Interestingly, only IgM9 and three of IgGs (IgG11, IgG14, and IgG19) demonstrated significant inhibition by one inhibitor (iodoacetamide). At the same time other IgG and IgM preparations were sensitive to two or three inhibitors (Table 2). For example, IgM10 and IgM11 was strongly suppressed by iodoacetamide and EDTA; IgM23 was sensitive to AEBSF and iodoacetamide; IgM4 and IgM8 showed strong or at least some inhibition of the activity by three inhibitors (iodoacetamide, EDTA, and pepstatin A), while IgM12 was sensitive to leupeptin instead of pepstatin A (Table 2). Surprisingly, three of ten preparations (IgM5, IgM7, and IgM13) could be significantly inhibited by four different inhibitors. Of these, iodoacetamide and EDTA were common inhibitors for all three IgM preparations, while two other inhibitors were different: pepstatin A and leupeptin for IgM5,

\*\*Sum of the effects of different compounds on the proteolytic activity

(leupeptin+Pepstatin+iodoacetamide + EDTA).

individual for every HIV-infected patient.

Inhibition, %\*

EDTA

Sum of effects\*\*

Number of prep.

leupeptin and AEBSF for IgM7, and pepstatin A and AEBSF for IgM13 (Table 2). Very comparable situations were observed for AIDS IgGs and IgMs (Table 2).

In principle it is possible that the pools of IgGs and IgMs from AIDS patients may be "cocktails" of Abz molecules, with each molecule possessing only one of four alternative types of proteolytic activity: serine-, acidic-, thiol-, or metal-dependent. Yet the effects of two, three, and four inhibitors of different protease types did not always add to 100% inhibition. Only in three of ten IgM preparations (IgM8, IgM9, and IgM11) this sum was less or comparable with 100% (76–114%), while for other seven IgMs it varied from 125% to 289% (Table 2). Three IgG preparations (IgG9, IgG10, and IgG13) demonstrated this sum from 249 to 255 % (Table 2). Since IgM9 had only thiol protease-like activity, and IgM8 and IgM11 could be significantly suppressed (94–99%) only with iodoacetamide but lost their activity only marginally in the presence of EDTA or pepstatin A (by 7–8 %), it is most likely that in these patients most of the Abz molecules possess only the thiol protease type of proteolytic activity (Table 2). However, since the proteolytic activity in seven of ten IgMs and five of ten IgGs was summarily suppressed by specific inhibitors of serine, acidic, metaldependent, and thiol proteases by more than 100% (110–289%, Table 2), it is possible that the immune system of HIV-infected patients produces anti-IN Abzs with a combined structure of the active center, carrying amino acid residues typical of different proteases. For example, we suggest that the pools of IgM4, IgM7, and IgM13 (209–289% of the summarized inhibition) contain IgM molecules with extremely complicated active centers containing structural elements of thiol and metal-dependent proteases, which may be additionally combined with structural elements of the active centers of acidic proteases (IgM4), serine proteases (IgM7), or both (IgM13). Similar suggestion is reason in the case of several AIDS IgGs (Table 2) (Baranova et al., 2009, 2010).

#### **5.2 pH optima diversity of AIDS abzymes**

Theoretically, a mammalian immune system can produce up to 106 variants of Abs against one antigen. An extreme diversity of RNase and DNase IgG and/or IgM Abzs from the sera of patients with MS and SLE and autoimmune prone MRL-lpr/lpr mice was observed (Baranovskii *et al.,* 1998, 2001; Andrievskaya *et al*., 2000, 2002; Kuznetsova *et al.,* 2007; Nevinsky *et al.*, 2003, 2005, 2010a, 2010b). It was shown that different patients (and animals) may have a relatively small or an extremely large pool of polyclonal nuclease Abzs containing different relative amounts of light chains of - and -types, demonstrating maximal activity at various optimal pHs, having a different net charge, activated or not by different metal ions, characterized by different substrate specificities. MS IgGs of all four subclasses (IgG1-IgG4) were catalytically active in the hydrolysis of hMBP (Legostaeva *et al*., 2010) and DNA (Parkhomenko *et al*., 2010).