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 demonstrates two marked pH optima at pH 7.5 and 8.5.

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 171

different individuals analyzed, but also other subfractions of Abs, the activity of which may be comparable with or less than that of the major subfraction. In addition, the number of *K*m and *V*max values, characterizing interaction of different monoclonal of polyclonal Abzs with their specific substrates, can significantly increase when they measured at several pH values

It was shown previously, that nuclease and protease Abzs from the sera of AI patients and animals are very heterogeneous in their affinity for cognate substrate and can be separated into many fractions by chromatography on affinity resins bearing immobilized substrate (Baranovskii et al., 2001; Andrievskaya et al., 2002; Semenov et al., 2004; Kuznetsova et al., 2007; Nevinsky et al., 1998, 2005, 2010a, 2010b). We have analyzed the affinity of AIDS pIgGs for human -casein by chromatography on casein-Sepharose (Odintsova et al., 2006b). Interestingly, when IgGs were eluted from casein-Sepharose by a KCl gradient (0–3 M), the protein (and casein-hydrolyzing activity) was distributed all over the chromatography profile. A similar result was obtained at AIDS IgGs chromatography on RT- and HSA-Sepharoses (Odintsova et al., 2006b). The data indicate for extreme affinity heterogeneity of casein-, RT-, and HSA-hydrolyzing abzymes to cognate protein substrates (Odintsova et al.,

We have subjected an equimolar mixture of pIgGs (and IgMs) from five AIDS patients to affinity chromatography on IN-Sepharose. Only 15±3 % of the total IgGs (Fig. 8A) and 173 % of the total IgMs (Fig. 8B) were bound to IN-Sepharose (Baranova et al., 2009, 2010). As we have shown previously, the fraction of Abzs with different catalytic activities including in the serum of AI patients usually does not exceed 0.1-5 % of total Igs (Nevinsky et al., 2005, 2010a, 2010b). Therefore, it was surprising that IN-Sepharose can bind up to 15-17 % of the total pIgGs and pIgMs. At the same time, IN is known as a very hydrophobic protein which can interact nonspecifically with different hydrophobic compounds including other proteins. Taking this into account we could suppose that immobilized IN binds anti-IN pIgGs and pIgMs in a specific manner, and interacts with some other IgGs and IgMs non-specifically.

A B

**RA, %**

**80**

**0**

(5 l of dialyzed eluate, total volume of the reaction mixture 10 l) corresponding to complete hydrolysis of 0.3 mg/ml IN after 16 h of incubation was taken for 100%.

Fig. 8. Affinity chromatography of the mixture of five individual AIDS pIgGs (A) and pIgMs (B) on IN-Sepharose: (), absorbance at 280 nm; the relative activity (RA) of IgGs and IgMs

**20**

**V**

**40**

**60**

**0 5 10 15 20 25**

**Fraction number**

**III**

**Gradient**

**4 3 2**

**3 M MgCl2**

**Gly-HCl, pH 2.6**

**IV**

**2 M MgCl2**

**3 M NaCl**

**0.0 0.2 0.4 0.6 0.8 1.0 1.2**

**Loading**

**I**

**1**

**0 5 10 15 20 25**

**2**

**II**

**Fraction number**

**Gradient**

**III**

**3 M NaCl 0.2 M NaCl Gly-HCl, pH 2.6 A280 RA, %**

**3 M MgCl2 2 M MgCl2**

**IV**

**3**

**4**

**V**

**5.3 Affinity and relative catalytic activity diversity of AIDS abzymes**

(Nevinsky, 2010b).

2006).

**0.2**

**0.4 0.6**

**I**

**1**

**II**

**0.2 M NaCl Loading**

**0.8**

**1.0**

**A280**

We have analyzed the pH dependencies of the initial rates of human casein hydrolysis by four individual AIDS IgGs. In contrast to all human proteases having one pronounced pH optimum (Horl et al., 1987; Rao et al., 1998), catalytic AIDS IgGs demonstrated high specific casein-hydrolyzing activity within a wide range of pH values (5.0–9.0) and the pH profile for each IgG was unique (Fig. 7B) (Odintsova et al., 2006).

It is well known that canonical mammalian, bacterial, and plant proteases, depending on their biological function, can have optimal pH values ranging from acidic (2.0) to neutral and alkaline (8-10) (Horl et al., 1987; Rao et al., 1998). We have measured the relative activity of AIDS IgGs and IgMs at pH from 3.0 to 10.5. In contrast to all human proteases, catalytic IgGs demonstrated high specific IN-hydrolyzing activity within a wide range of pH values (3.0–10).

Fig. 7. pH dependences of the relative DNase (A), casein-hydrolyzing (B) activity of IgGs, and IN-hydrolyzing activity (RA) of individual pIgGs (C) and pIgMs (D) from the sera of several different AIDS patients (number of Ab correspond to number of patient). The average error in the initial rate determination at each pH from two experiments did not exceed 7-10 %.

The pH profile for each IgG and IgM was unique; each preparation demonstrated from three to seven brightly expressed optima at different pH from 3 to 11 (Fig. 7). Taking into account the effective hydrolysis of IN at pH 3.0, one cannot exclude that human immune system of AIDS patients could in principle produce IgGs and IgMs with a proteolytic activity similar to that of stomach acidic proteases. The above results clearly demonstrate that pIgGs and pIgMs from individual AIDS patients can consist of different sets of catalytic Ab subfractions demonstrating quite distinct enzymic properties in the hydrolysis of DNA, human casein, and integrase.

Overall, a pool of many auto-Abs may contain very different monoclonal Abzs with various pH optima. It should be mentioned that the RAs of Abzs from patients with different AI and viral diseases are usually compared at one fixed pH, in which all samples are more or less active. Changing the reaction pH, one can reveal not only the major fraction of Abzs in

We have analyzed the pH dependencies of the initial rates of human casein hydrolysis by four individual AIDS IgGs. In contrast to all human proteases having one pronounced pH optimum (Horl et al., 1987; Rao et al., 1998), catalytic AIDS IgGs demonstrated high specific casein-hydrolyzing activity within a wide range of pH values (5.0–9.0) and the pH profile

It is well known that canonical mammalian, bacterial, and plant proteases, depending on their biological function, can have optimal pH values ranging from acidic (2.0) to neutral and alkaline (8-10) (Horl et al., 1987; Rao et al., 1998). We have measured the relative activity of AIDS IgGs and IgMs at pH from 3.0 to 10.5. In contrast to all human proteases, catalytic IgGs demonstrated high specific IN-hydrolyzing activity within a wide range of pH values

for each IgG was unique (Fig. 7B) (Odintsova et al., 2006).

**3 4 5 6 7 8 9 10 11 12**

**pH**

 **IgG3**

Fig. 7. pH dependences of the relative DNase (A), casein-hydrolyzing (B) activity of IgGs, and IN-hydrolyzing activity (RA) of individual pIgGs (C) and pIgMs (D) from the sera of several different AIDS patients (number of Ab correspond to number of patient). The average error in the initial rate determination at each pH from two experiments did not

The pH profile for each IgG and IgM was unique; each preparation demonstrated from three to seven brightly expressed optima at different pH from 3 to 11 (Fig. 7). Taking into account the effective hydrolysis of IN at pH 3.0, one cannot exclude that human immune system of AIDS patients could in principle produce IgGs and IgMs with a proteolytic activity similar to that of stomach acidic proteases. The above results clearly demonstrate that pIgGs and pIgMs from individual AIDS patients can consist of different sets of catalytic Ab subfractions demonstrating quite distinct enzymic properties in the hydrolysis of DNA,

Overall, a pool of many auto-Abs may contain very different monoclonal Abzs with various pH optima. It should be mentioned that the RAs of Abzs from patients with different AI and viral diseases are usually compared at one fixed pH, in which all samples are more or less active. Changing the reaction pH, one can reveal not only the major fraction of Abzs in

**RA, %**

**40**

**60**

**80**

**100**

**RA, %**

**IgM1**

**4.8 6.0 7.2 8.4 9.6**

**56789**

**IgG16**

**IgG8 IgG14 IgG4**

 **IgM2**

 **IgM4**

**D**

**B**

**pH**

 **IgM3**

**pH**

 **IgG1**

**IgG7 IgG6**

**pH**

**IgG5**

exceed 7-10 %.

human casein, and integrase.

**RA, %**

 **IgG2**

**C**

**A**

**56789**

(3.0–10).

different individuals analyzed, but also other subfractions of Abs, the activity of which may be comparable with or less than that of the major subfraction. In addition, the number of *K*m and *V*max values, characterizing interaction of different monoclonal of polyclonal Abzs with their specific substrates, can significantly increase when they measured at several pH values (Nevinsky, 2010b).

#### **5.3 Affinity and relative catalytic activity diversity of AIDS abzymes**

It was shown previously, that nuclease and protease Abzs from the sera of AI patients and animals are very heterogeneous in their affinity for cognate substrate and can be separated into many fractions by chromatography on affinity resins bearing immobilized substrate (Baranovskii et al., 2001; Andrievskaya et al., 2002; Semenov et al., 2004; Kuznetsova et al., 2007; Nevinsky et al., 1998, 2005, 2010a, 2010b). We have analyzed the affinity of AIDS pIgGs for human -casein by chromatography on casein-Sepharose (Odintsova et al., 2006b). Interestingly, when IgGs were eluted from casein-Sepharose by a KCl gradient (0–3 M), the protein (and casein-hydrolyzing activity) was distributed all over the chromatography profile. A similar result was obtained at AIDS IgGs chromatography on RT- and HSA-Sepharoses (Odintsova et al., 2006b). The data indicate for extreme affinity heterogeneity of casein-, RT-, and HSA-hydrolyzing abzymes to cognate protein substrates (Odintsova et al., 2006).

We have subjected an equimolar mixture of pIgGs (and IgMs) from five AIDS patients to affinity chromatography on IN-Sepharose. Only 15±3 % of the total IgGs (Fig. 8A) and 173 % of the total IgMs (Fig. 8B) were bound to IN-Sepharose (Baranova et al., 2009, 2010).

As we have shown previously, the fraction of Abzs with different catalytic activities including in the serum of AI patients usually does not exceed 0.1-5 % of total Igs (Nevinsky et al., 2005, 2010a, 2010b). Therefore, it was surprising that IN-Sepharose can bind up to 15-17 % of the total pIgGs and pIgMs. At the same time, IN is known as a very hydrophobic protein which can interact nonspecifically with different hydrophobic compounds including other proteins. Taking this into account we could suppose that immobilized IN binds anti-IN pIgGs and pIgMs in a specific manner, and interacts with some other IgGs and IgMs non-specifically.

Fig. 8. Affinity chromatography of the mixture of five individual AIDS pIgGs (A) and pIgMs (B) on IN-Sepharose: (), absorbance at 280 nm; the relative activity (RA) of IgGs and IgMs (5 l of dialyzed eluate, total volume of the reaction mixture 10 l) corresponding to complete hydrolysis of 0.3 mg/ml IN after 16 h of incubation was taken for 100%.

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 173

**12-18 M**

**V, M IN / 17 h**

Fig. 9. The dependencies of the initial rates of IN hydrolysis upon the IN concentration in the reaction catalyzed by non-separated by affinity chromatography two individual pIgMs (A) and two pIgGs (B) from different patients in coordinates V *vs* [S]. IgM12, IgM32, IgG15, and IgG32 were used in different concentrations. Arrows show different hyperbolic

**0 10 20 30 40 50 60 70**

**[IN], M**

**30-50 M**

**B**

**-0,02 -0,01 0,00 0,01 0,02 0,03 0,04 0,05**

**[IN], M**

**IgG**

**IgM**

**1 / V, min / M IN**

Fig. 10. The dependencies of the initial rates of IN hydrolysis upon the IN concentration in the reaction catalyzed by fractions of pIgGmix and pIgMmix purified on IN-Sepharose (Fig. 8; fractions number 2; IgGload and IgMload) in coordinates V vs [S] (A). Determination of the *Km*  for IN and *Vmax* values using the Lineweaver-Burk plot in the case of the same IgGload and

fraction 24 eluted with an acidic buffer) demonstrated 3– and 10-fold respectively higher affinity than that for pIgMload fraction (130± 30 M). The *Km* and *kcat* values corresponding to fractionated pIgGs and pIgMs agree with the relative contents and specific activities of the major Ab subfractions isolated by affinity chromatography (Fig. 8). On overall, the above data demonstrate extreme diversity of HIV IgGs and IgMs in their affinity to IN and in the

Affinity chromatography of DNase and RNase Abs from AI patients and animals (Baranovskii et al., 2001; Andrievskaya et al., 2002; Kuznetsova et al., 2007), healthy rabbits immunized with DNA, RNA, DNase I, DNase II, pancreatic RNase (Krasnorutskii et al.

**IgG32**

**80-100 M**

**IgG15**

**0 5 10 15 20 25 30 35**

**15-20 M**

**A**

**[IN], M**

**0 20 40 60 80 100 120 140 0,00**

**[IN], M**

**IgM32**

**30-50 M**

**IgM12**

fragments of complicated curves corresponding to the total dependencies.

**IgG**

**A IgM B**

**0**

**0,02 0,04 0,06 0,08 0,10 0,12 0,14**

**V, M IN / min**

IgMload (B).

relative *kcat* values (Figs 8-10).

**2**

**5-10 M**

**V, M IN / 8 h**

**4**

**6**

The pIgG and IgM fractions of the first peak (83-85% of protein loaded on IN-Sepharose) with a very low affinity for IN possess high IN-hydrolyzing activity (peak 1, Fig. 8). As it was shown previously non-separated on affinity resins pIgGs and IgMs from AIDS patients contain small subfractions hydrolyzing specifically not only HIV IN (Baranova et al., 2009, 2010), but also HIV RT, HSA and human casein (Odintsova et al., 2006). However, the fractions of pIgGs and pIgMs having high affinity to IN-Sepharose and eluted from this sorbent with different concentrations of NaCl, MgCl2 and acidic buffer (protein peaks 2-4, activity peaks II-V; Fig. 8) hydrolyzed only IN. Thus, IgGs and IgMs with IN-independent activities do not have affinity for IN-Sepharose, but some other Abs can be bound with IN non-specifically.

The total IN-hydrolyzing activities of pIgGs and pIgMs were distributed all over the chromatography profiles and in the case of both Abs five peaks of IN-hydrolyzing activity (I – V, Fig. 8) were brightly expressed. The data obtained are indicative of extreme heterogeneity of IN-hydrolyzing pIgGs and pIgMs in their affinity to IN.

When Abzs are highly heterogeneous, the dependence of *V* on the substrate concentration for non-fractionated Abs may appear inconsistent with simple Michaelis-Menten kinetics and may be described by a sum of several hyperbolic curves corresponding to different Ab subfractions. However, the contribution of some subfractions to the total curve may be small, or they may have comparable *Km* and *V*max (*kcat*) values. As a rule, only when significant differences (≥ 5-fold) exist between the *Km* and *V*max values for different Ab subfractions it is possible to determine these parameters characterizing individual subfractions of polyclonal Abz from the aggregated initial rate curves.

First, we have measured the *Km* and *V*max values in the reaction of IN hydrolysis using two individual preparations of pIgGs and pIgMs not fractionated on IN-Sepharose. The initial rate data obtained for these Abs at the increasing IN concentration were inconsistent with the Michaelis-Menten kinetics and the dependences corresponded to at least three or four hyperbolic curves with several segments reflecting different *Km* values, which were approximately in the ranges of 5-10, 15-20, 30-50, and higher than 70-100 M (Fig. 9). Similar situation was observed for the mixtures of equal amounts of electrophoretically homogeneous IgGs (pIgGmix) and IgMs (pIgMmix) from the sera of five AIDs patients.

For more detailed analysis of *Km* and *kcat* values characterizing different Ab fractions within total pool of Abzs (pIgGmix and pIgMmix) we have analyzed several individual pIgGmix and pIgMmix fractions eluted from IN-Sepharose (Fig. 8). First, we have measured the *Km* and *V*max values in the reaction catalyzed by IgGmix (IgGload) and IgMmix (IgMload) corresponding to the second fraction eluted under loading of Abs on IN-Sepharose (Fig. 8). The dependencies of V/[S] (hyperbolic curves; Fig. 10A) and 1/V *vs* 1/[S] (Fig. 10B) demonstrated virtually normal Michaelis-Menten character for second fractions of pIgGs and pIgMs. The *Km* and *kcat* for IgGload (156± 40 M; 0.3±0.1 min-1) and IgMload (130± 30 M; 2.0±0.4 min-1) were determined. The V/[S] and 1/V *vs* 1/[S] dependences for individual fractions of pIgGs and pIgMs eluted from IN-Sepharose in gradient of NaCl concentration (IgGsalt and IgMsalt) and by acidic buffer (IgGacid and IgMacid) had also typical Michaelis-Menten character.

The affinity of pIgGs for IN (in terms of *Km* values) increased with the increase of their affinity to IN-Sepharose; for IgGsalt (*Km* = 44±4.0 M) corresponding to fraction 8 (eluted with the salt) and IgGacid (*Km* = 14±1.0 M) corresponding to fraction 24 (eluted with an acidic buffer) the affinity was 3.5– and 11-fold respectively higher than that for IgGload (156±40 M). Similar situation was observed for the separated individual fractions of IgMmix (Fig. 8B); pIgMsolid (*Km* =43±4.0 M; fraction 10 eluted with NaCl) and pIgMacid (*Km* = 12.8±1.0 M;

The pIgG and IgM fractions of the first peak (83-85% of protein loaded on IN-Sepharose) with a very low affinity for IN possess high IN-hydrolyzing activity (peak 1, Fig. 8). As it was shown previously non-separated on affinity resins pIgGs and IgMs from AIDS patients contain small subfractions hydrolyzing specifically not only HIV IN (Baranova et al., 2009, 2010), but also HIV RT, HSA and human casein (Odintsova et al., 2006). However, the fractions of pIgGs and pIgMs having high affinity to IN-Sepharose and eluted from this sorbent with different concentrations of NaCl, MgCl2 and acidic buffer (protein peaks 2-4, activity peaks II-V; Fig. 8) hydrolyzed only IN. Thus, IgGs and IgMs with IN-independent activities do not have affinity for IN-Sepharose, but some other Abs can be bound with IN non-specifically. The total IN-hydrolyzing activities of pIgGs and pIgMs were distributed all over the chromatography profiles and in the case of both Abs five peaks of IN-hydrolyzing activity (I – V, Fig. 8) were brightly expressed. The data obtained are indicative of extreme

When Abzs are highly heterogeneous, the dependence of *V* on the substrate concentration for non-fractionated Abs may appear inconsistent with simple Michaelis-Menten kinetics and may be described by a sum of several hyperbolic curves corresponding to different Ab subfractions. However, the contribution of some subfractions to the total curve may be small, or they may have comparable *Km* and *V*max (*kcat*) values. As a rule, only when significant differences (≥ 5-fold) exist between the *Km* and *V*max values for different Ab subfractions it is possible to determine these parameters characterizing individual

First, we have measured the *Km* and *V*max values in the reaction of IN hydrolysis using two individual preparations of pIgGs and pIgMs not fractionated on IN-Sepharose. The initial rate data obtained for these Abs at the increasing IN concentration were inconsistent with the Michaelis-Menten kinetics and the dependences corresponded to at least three or four hyperbolic curves with several segments reflecting different *Km* values, which were approximately in the ranges of 5-10, 15-20, 30-50, and higher than 70-100 M (Fig. 9). Similar situation was observed for the mixtures of equal amounts of electrophoretically

homogeneous IgGs (pIgGmix) and IgMs (pIgMmix) from the sera of five AIDs patients.

For more detailed analysis of *Km* and *kcat* values characterizing different Ab fractions within total pool of Abzs (pIgGmix and pIgMmix) we have analyzed several individual pIgGmix and pIgMmix fractions eluted from IN-Sepharose (Fig. 8). First, we have measured the *Km* and *V*max values in the reaction catalyzed by IgGmix (IgGload) and IgMmix (IgMload) corresponding to the second fraction eluted under loading of Abs on IN-Sepharose (Fig. 8). The dependencies of V/[S] (hyperbolic curves; Fig. 10A) and 1/V *vs* 1/[S] (Fig. 10B) demonstrated virtually normal Michaelis-Menten character for second fractions of pIgGs and pIgMs. The *Km* and *kcat* for IgGload (156± 40 M; 0.3±0.1 min-1) and IgMload (130± 30 M; 2.0±0.4 min-1) were determined. The V/[S] and 1/V *vs* 1/[S] dependences for individual fractions of pIgGs and pIgMs eluted from IN-Sepharose in gradient of NaCl concentration (IgGsalt and IgMsalt) and by acidic buffer

The affinity of pIgGs for IN (in terms of *Km* values) increased with the increase of their affinity to IN-Sepharose; for IgGsalt (*Km* = 44±4.0 M) corresponding to fraction 8 (eluted with the salt) and IgGacid (*Km* = 14±1.0 M) corresponding to fraction 24 (eluted with an acidic buffer) the affinity was 3.5– and 11-fold respectively higher than that for IgGload (156±40 M). Similar situation was observed for the separated individual fractions of IgMmix (Fig. 8B); pIgMsolid (*Km* =43±4.0 M; fraction 10 eluted with NaCl) and pIgMacid (*Km* = 12.8±1.0 M;

heterogeneity of IN-hydrolyzing pIgGs and pIgMs in their affinity to IN.

subfractions of polyclonal Abz from the aggregated initial rate curves.

(IgGacid and IgMacid) had also typical Michaelis-Menten character.

Fig. 9. The dependencies of the initial rates of IN hydrolysis upon the IN concentration in the reaction catalyzed by non-separated by affinity chromatography two individual pIgMs (A) and two pIgGs (B) from different patients in coordinates V *vs* [S]. IgM12, IgM32, IgG15, and IgG32 were used in different concentrations. Arrows show different hyperbolic fragments of complicated curves corresponding to the total dependencies.

Fig. 10. The dependencies of the initial rates of IN hydrolysis upon the IN concentration in the reaction catalyzed by fractions of pIgGmix and pIgMmix purified on IN-Sepharose (Fig. 8; fractions number 2; IgGload and IgMload) in coordinates V vs [S] (A). Determination of the *Km*  for IN and *Vmax* values using the Lineweaver-Burk plot in the case of the same IgGload and IgMload (B).

fraction 24 eluted with an acidic buffer) demonstrated 3– and 10-fold respectively higher affinity than that for pIgMload fraction (130± 30 M). The *Km* and *kcat* values corresponding to fractionated pIgGs and pIgMs agree with the relative contents and specific activities of the major Ab subfractions isolated by affinity chromatography (Fig. 8). On overall, the above data demonstrate extreme diversity of HIV IgGs and IgMs in their affinity to IN and in the relative *kcat* values (Figs 8-10).

Affinity chromatography of DNase and RNase Abs from AI patients and animals (Baranovskii et al., 2001; Andrievskaya et al., 2002; Kuznetsova et al., 2007), healthy rabbits immunized with DNA, RNA, DNase I, DNase II, pancreatic RNase (Krasnorutskii et al.

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 175

vary in the range of 0.001–40 min–1 (Gololobov et al., 1995; Kalaga et al., 1995; Nevinsky et al., 2005, 2010a, 20010b). There are only several exceptions. For example, the specific activity of RNase IgGs from AI patients was about 1-20% of that for RNase A and of six known human sera RNases, while poly(A) was hydrolyzed by Abzs 2-10-fold faster than by RNase A, one of the most active RNases known (Buneva et al., 1994; Baranovskii et al., 1997, 1998; Vlasov et al., 1998). At the same time, the specific activity of homogeneous Abzs of several SLE and MS patients was about 40-400% of that for RNase A (Baranovskii et al., 1998). In addition, the specific nucleotide-hydrolyzing activities of mouse polyclonal IgGs is 1–4 orders of magnitude higher that those of known natural Abzs (Andryushkova et al., 2009). The *kcat* values for AIDS pIgGs hydrolyzing scDNA ((2.1-29.6)×10-2 min-1), human casein (2.0×10-2 min-1), HSA (3.6×10-5 min-1), and HIV RT (2.2×10-3 min-1), as well IgGs (0.3-2.9 min-1) and IgMs (2.0-6.4 min-1) purified on IN-Sepharose (Table 2) in the IN hydrolysis were comparable with those for known Abzs (Odintsova et al., 2006a, 2006b; Baranova et al., 2009,

Currently there are no methods that can efficiently separate Abzs from catalytically inactive Abs against the same antigen. In addition, as it was shown above, IN-Sepharose interact not only with anti-IN IgMs and IgGs but bind non-specifically some other Abs. Even partial purification of IgGs and IgMs on IN-Sepharose (or other specific affinity resins) leads to significant increase in the *kcat* value for IN and other substrates hydrolysis. Since the specific activities in all cases were calculated using the total concentration of purified pIgGs and pIgMs and affinity chromatography on IN-Sepharose (and other affinity sorbents) cannot separate catalytically active and inactive anti-IN Abs, the specific IN-hydrolyzing activities of the individual monoclonal subfractions in the pIgG and pIgM pools may be higher than those of non-fractionated or partially fractionated Abs. It should be mentioned that specific activities of some Abzs are often significantly lower than those for canonical enzymes with the same catalytic activities. However, it is impossible to dismiss the RAs of Abs from patients with AI and viral infection as biologically inessential since they are comparable with those for many canonical and very important enzymes, for example, restriction endonucleases and repair enzymes (Gololobov et al., 1995; Nevinsky et al., 2005, 2010, 2010). Thus, IN-hydrolyzing IgGs and IgMs from HIV-infected patients are very heterogeneous in their affinity to IN-Sepharose, demonstrate different *Km* and *Vmax* values and different subfractions of Abzs can hydrolyze various substrates at pH from 3 to 10. In addition, in contrast to other Abzs with proteolytic activity they can possess for different types of

proteolytic activities: thiol-, metal-dependent, serine- and acidic-like.

**6.1 Casein hydrolysis by AIDS abzymes**

**6. Peculiarities of protein hydrolysis by AIDS abzymes and canonical**

syndrome, polymyositis, AI myocarditis, etc. (Amino *et al.*, 1982; Freeman *et al.*, 1986).

Casein hydrolyzing Abzs was found not only in the sera of HIV-infected patients (Odintsova et al., 2006b) but also in the milk of lactating women (Odintsova et al., 2005; 2011). At the first glance, no obvious immunizing factors could be found in clinically healthy pregnant and lactating women. However, pregnancy could "activate" or "trigger" autoimmune-like manifestations in clinically healthy women, and a sharp exacerbation of AI reactions can occur in some cases soon after childbirth (Amino *et al.*, 1982; Freeman *et al.*, 1986). Postnatal AI pathologies arise sometimes, including SLE, Hashimoto's thyroiditis, phospholipids

2010).

**proteases**

2008a, 2008b, 2008c, 2008d, 2009) as well as AI Abs with proteolytic and other activities (Nevinsky et al, 1998; Semenov et al., 2004; Legostaeva et al., 2010) on resins bearing immobilized specific substrates using elution of Abs with different concentration of NaCl and an acidic buffer always leads to separation of Abs into many Abz subfractions with different affinity to immobilized substrate. In addition, the affinity of separated fractions for immobilized substrate increased gradually with the increase in eluting NaCl concentration, but the *Km* (and *Vmax*) values corresponding to each fraction eluted from affinity sorbent are individual for every patient analyzed. It means, that the apparent number of monoclonal Abzs with different catalytic properties within the polyclonal Abs pool may be significantly underestimated since it is impossible to separate Abzs with comparable affinities for a specific substrate or to distinguish monoclonal Abzs with similar kinetic parameters. Some minor monoclonal Abzs with a relatively high activity and even major Abzs with low activity may be hidden by major Abzs with high activity. As a consequence, the *Km* (and *Vmax*) values determined using Abzs not fractionated on affinity sorbents more often characterize the interaction of substrates with a major fraction of Abzs with the maximal content and highest relative enzymatic activity at condition used. At the same time, these characteristic are very useful for comparisons of Abs with different substrate specificities and from patients with different diseases. For example, it was shown that affinity of specific major fractions of AIDS IgGs non-fractionated on affinity resins to HSA (*Km* = (1.8 ± 0.6)×10-8 M, *kcat* = (3.6± 1.1)×10-5 min-1) is 270-290-fold higher than to HIV RT (*Km* = (4.9 ± 0.5)×10-6 M, *kcat* = (2.2± 0.2)×10-3 min-1) and to -casein (*Km* = (5.3 ± 0.5)·10-6 M, *kcat* = (2.0± 0.2)×10-2 min-1) (Odintsova et al., 2006). At the same time, the relative rate of -casein hydrolysis was 9- and 550-fold higher than that for HIV RT and HSA, respectively. The *Km* for casein ((7.3±1.2)×10-6 M; *k*cat = 0.75±0.05 min-1) estimated in the reaction catalyzed by polyclonal sIgAs from human milk (Odintsova et al., 2005) is comparable with that for IgGs from AIDS patients, while the *kcat* is ~38-fold higher. This difference is most likely due to a higher content of anticasein proteolytic Abzs in human milk in comparison with blood of AIDS patients.

The affinity of AIDS pIgGs hydrolyzing HIV RT, human casein, and HSA (*Km* = 0.018 – 5.3 M; see above) as well as IN-Sepharose-purified AIDS pIgGs and pIgMs for IN (*Km* = 12.8- 156 M) in terms of *Km* values is comparable with typical affinities (*Km* = 0.038–7.3 M) (Paul et al., 1989; Kalaga et al., 1995; Legostaeva et al., 2010; Nevinsky et al., 2005, 2010a, 2010b) of Abzs hydrolyzing different proteins.

The *Km* (and *kcat*) values for plasmid scDNA in the reaction catalyzed by two individual nonfractionated AIDS IgG preparations were determined (Odintsova et al., 2006a). In the case of one of two preparations analyzed, the initial rate of DNA hydrolysis increases with increase in DNA concentration according to the Michaelis–Menten kinetics and only one pair of *K*m (53 ± 9 nM) and *kcat* ((2.1 ± 0.1)×10-2 min-1) was observed. For the second pIgG preparation two pairs of *K*m (2.6 ± 0.1 and 4.4 ± 0.7 nM) and *kcat* values ((6.7 ± 0.1)×10-2 and (29.6 ± 5.0)×10- 2 min-1) were revealed*.* Thus, the affinity the scDNA substrate for AIDS IgGs varied (in terms of *Km* values) in the range 2.6–53 nM, which correspond to typical *Kd* values for Ab– antigen interactions and is about 3–4 orders of magnitude higher than affinity of scDNA for DNase I (*KM* = 46–58 M) (Gololobov et al., 1995). These *Km* values for scDNA are comparable with the *Km* for plasmid DNA (43 nM) reported previously for IgG from SLE patients (Gololobov et al., 1995).

The catalysis mediated by artificial Abzs against reaction transition states is usually characterized by relatively low reaction rates: *kcat* values are 102–106-fold lower than for canonical enzymes (Keinan, 2005). The known *kcat* values for natural Abzs from AI patients

2008a, 2008b, 2008c, 2008d, 2009) as well as AI Abs with proteolytic and other activities (Nevinsky et al, 1998; Semenov et al., 2004; Legostaeva et al., 2010) on resins bearing immobilized specific substrates using elution of Abs with different concentration of NaCl and an acidic buffer always leads to separation of Abs into many Abz subfractions with different affinity to immobilized substrate. In addition, the affinity of separated fractions for immobilized substrate increased gradually with the increase in eluting NaCl concentration, but the *Km* (and *Vmax*) values corresponding to each fraction eluted from affinity sorbent are individual for every patient analyzed. It means, that the apparent number of monoclonal Abzs with different catalytic properties within the polyclonal Abs pool may be significantly underestimated since it is impossible to separate Abzs with comparable affinities for a specific substrate or to distinguish monoclonal Abzs with similar kinetic parameters. Some minor monoclonal Abzs with a relatively high activity and even major Abzs with low activity may be hidden by major Abzs with high activity. As a consequence, the *Km* (and *Vmax*) values determined using Abzs not fractionated on affinity sorbents more often characterize the interaction of substrates with a major fraction of Abzs with the maximal content and highest relative enzymatic activity at condition used. At the same time, these characteristic are very useful for comparisons of Abs with different substrate specificities and from patients with different diseases. For example, it was shown that affinity of specific major fractions of AIDS IgGs non-fractionated on affinity resins to HSA (*Km* = (1.8 ± 0.6)×10-8 M, *kcat* = (3.6± 1.1)×10-5 min-1) is 270-290-fold higher than to HIV RT (*Km* = (4.9 ± 0.5)×10-6 M, *kcat* = (2.2± 0.2)×10-3 min-1) and to -casein (*Km* = (5.3 ± 0.5)·10-6 M, *kcat* = (2.0± 0.2)×10-2 min-1) (Odintsova et al., 2006). At the same time, the relative rate of -casein hydrolysis was 9- and 550-fold higher than that for HIV RT and HSA, respectively. The *Km* for casein ((7.3±1.2)×10-6 M; *k*cat = 0.75±0.05 min-1) estimated in the reaction catalyzed by polyclonal sIgAs from human milk (Odintsova et al., 2005) is comparable with that for IgGs from AIDS patients, while the *kcat* is ~38-fold higher. This difference is most likely due to a higher content of anti-

casein proteolytic Abzs in human milk in comparison with blood of AIDS patients.

Abzs hydrolyzing different proteins.

patients (Gololobov et al., 1995).

The affinity of AIDS pIgGs hydrolyzing HIV RT, human casein, and HSA (*Km* = 0.018 – 5.3 M; see above) as well as IN-Sepharose-purified AIDS pIgGs and pIgMs for IN (*Km* = 12.8- 156 M) in terms of *Km* values is comparable with typical affinities (*Km* = 0.038–7.3 M) (Paul et al., 1989; Kalaga et al., 1995; Legostaeva et al., 2010; Nevinsky et al., 2005, 2010a, 2010b) of

The *Km* (and *kcat*) values for plasmid scDNA in the reaction catalyzed by two individual nonfractionated AIDS IgG preparations were determined (Odintsova et al., 2006a). In the case of one of two preparations analyzed, the initial rate of DNA hydrolysis increases with increase in DNA concentration according to the Michaelis–Menten kinetics and only one pair of *K*m (53 ± 9 nM) and *kcat* ((2.1 ± 0.1)×10-2 min-1) was observed. For the second pIgG preparation two pairs of *K*m (2.6 ± 0.1 and 4.4 ± 0.7 nM) and *kcat* values ((6.7 ± 0.1)×10-2 and (29.6 ± 5.0)×10- 2 min-1) were revealed*.* Thus, the affinity the scDNA substrate for AIDS IgGs varied (in terms of *Km* values) in the range 2.6–53 nM, which correspond to typical *Kd* values for Ab– antigen interactions and is about 3–4 orders of magnitude higher than affinity of scDNA for DNase I (*KM* = 46–58 M) (Gololobov et al., 1995). These *Km* values for scDNA are comparable with the *Km* for plasmid DNA (43 nM) reported previously for IgG from SLE

The catalysis mediated by artificial Abzs against reaction transition states is usually characterized by relatively low reaction rates: *kcat* values are 102–106-fold lower than for canonical enzymes (Keinan, 2005). The known *kcat* values for natural Abzs from AI patients vary in the range of 0.001–40 min–1 (Gololobov et al., 1995; Kalaga et al., 1995; Nevinsky et al., 2005, 2010a, 20010b). There are only several exceptions. For example, the specific activity of RNase IgGs from AI patients was about 1-20% of that for RNase A and of six known human sera RNases, while poly(A) was hydrolyzed by Abzs 2-10-fold faster than by RNase A, one of the most active RNases known (Buneva et al., 1994; Baranovskii et al., 1997, 1998; Vlasov et al., 1998). At the same time, the specific activity of homogeneous Abzs of several SLE and MS patients was about 40-400% of that for RNase A (Baranovskii et al., 1998). In addition, the specific nucleotide-hydrolyzing activities of mouse polyclonal IgGs is 1–4 orders of magnitude higher that those of known natural Abzs (Andryushkova et al., 2009). The *kcat* values for AIDS pIgGs hydrolyzing scDNA ((2.1-29.6)×10-2 min-1), human casein (2.0×10-2 min-1), HSA (3.6×10-5 min-1), and HIV RT (2.2×10-3 min-1), as well IgGs (0.3-2.9 min-1) and IgMs (2.0-6.4 min-1) purified on IN-Sepharose (Table 2) in the IN hydrolysis were comparable with those for known Abzs (Odintsova et al., 2006a, 2006b; Baranova et al., 2009, 2010).

Currently there are no methods that can efficiently separate Abzs from catalytically inactive Abs against the same antigen. In addition, as it was shown above, IN-Sepharose interact not only with anti-IN IgMs and IgGs but bind non-specifically some other Abs. Even partial purification of IgGs and IgMs on IN-Sepharose (or other specific affinity resins) leads to significant increase in the *kcat* value for IN and other substrates hydrolysis. Since the specific activities in all cases were calculated using the total concentration of purified pIgGs and pIgMs and affinity chromatography on IN-Sepharose (and other affinity sorbents) cannot separate catalytically active and inactive anti-IN Abs, the specific IN-hydrolyzing activities of the individual monoclonal subfractions in the pIgG and pIgM pools may be higher than those of non-fractionated or partially fractionated Abs. It should be mentioned that specific activities of some Abzs are often significantly lower than those for canonical enzymes with the same catalytic activities. However, it is impossible to dismiss the RAs of Abs from patients with AI and viral infection as biologically inessential since they are comparable with those for many canonical and very important enzymes, for example, restriction endonucleases and repair enzymes (Gololobov et al., 1995; Nevinsky et al., 2005, 2010, 2010). Thus, IN-hydrolyzing IgGs and IgMs from HIV-infected patients are very heterogeneous in their affinity to IN-Sepharose, demonstrate different *Km* and *Vmax* values and different subfractions of Abzs can hydrolyze various substrates at pH from 3 to 10. In addition, in contrast to other Abzs with proteolytic activity they can possess for different types of proteolytic activities: thiol-, metal-dependent, serine- and acidic-like.

#### **6. Peculiarities of protein hydrolysis by AIDS abzymes and canonical proteases**

#### **6.1 Casein hydrolysis by AIDS abzymes**

Casein hydrolyzing Abzs was found not only in the sera of HIV-infected patients (Odintsova et al., 2006b) but also in the milk of lactating women (Odintsova et al., 2005; 2011). At the first glance, no obvious immunizing factors could be found in clinically healthy pregnant and lactating women. However, pregnancy could "activate" or "trigger" autoimmune-like manifestations in clinically healthy women, and a sharp exacerbation of AI reactions can occur in some cases soon after childbirth (Amino *et al.*, 1982; Freeman *et al.*, 1986). Postnatal AI pathologies arise sometimes, including SLE, Hashimoto's thyroiditis, phospholipids syndrome, polymyositis, AI myocarditis, etc. (Amino *et al.*, 1982; Freeman *et al.*, 1986).

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 177

HIV-1 integrase catalyzes insertion of a DNA copy of the viral genome into the host genome (Asante-Appiah & Skalka, 1999)*.* Therefore IN, together with RT and protease, is the main

Specific interactions between HIV IN and long terminal repeats are required for insertion of viral DNA into the host genome. The use of a method based on stepwise increase in ligand complexity allowed an estimation of the relative contributions of each nucleotide from oligonucleotides (ODNs) to the total affinity for IN (Bugreev et al., 2003). It was shown that IN interacts with ODNs through superposition of weak contacts with their bases and, more importantly, with the internucleotide phosphate groups. Formation of the IN complex with specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the *kcat* term; the rate of 3'-processin reaction is increased by more than 5 orders of magnitude upon transition from non-specific to specific oligonucleotides (Bugreev et al.,

In solution, HIV-1 IN exists in a dynamic equilibrium of monomers, dimers, tetramers and high-order oligomers (Deprez et al., 2000). We have recently analyzed the activity of different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking and shown that these forms do not share the same *in vitro* catalytic properties (Faure et al., 2005). While monomers were inactive for all specific IN activities, dimers were able to catalyze the 3'-processing and the insertion of only one LTR into a short target DNA. In contrast, a tetramer of IN catalyzed the full-site integration of the two viral

To characterize the influence of the determinants of DNA substrate specificity on the oligomerization status of IN, the small-angle X-ray scattering technique was used (Baranova et al., 2007). Under special conditions in the absence of ODNs IN existed only as monomers. IN preincubation with specific ODNs led mainly to formation of dimers, the relative amount of which correlated well with the increase in the enzyme activity. Under these conditions, tetramers were scarce. Nonspecific ODNs stimulated formation of catalytically inactive dimers and tetramers. Complexes of monomeric, dimeric and tetrameric forms of IN with specific and nonspecific ODNs had varying radii of gyration (Rg), suggesting that the specific sequence-dependent formation of IN tetramers occurs by dimerization of two dimers of different structure. From the data it was concluded that the DNA-induced oligomerization of HIV-1 IN is of extreme importance to provide substrate specificity and to

It is known that a formation of multiple contacts between the same or various subunits of oligomeric enzymes is usually provided by multiple hydrophobic and electrostatic contacts and hydrogen bonds. A similar situation was observed for the dimeric forms of HIV-1 IN by X-ray crystallography (for review see Wlodawer 1999; Chiu & Davies, 2004 ). Analysis of effects of specific and nonspecific ODNs on the rate of IN proteolysis by chymotrypsin, trypsin, and proteinase K can provide useful information concerning a possible decrease in the accessibility of aromatic and positively charged amino acid residues after an enzyme binds its substrates, changes its conformation, or forms contacts between its subunits. It was interesting to compare the effect of different ODNs on the cleavage of IN by Abzs and

**6.2 Integrase hydrolysis by AIDS abzymes**

important target of anti-HIV drugs.

LTR ends into a target DNA.

increase the enzyme activity (Baranova et al., 2007).

**6.2.2 Effect of DNA on the integrase hydrolysis by different proteases**

2003).

**6.2.1 Specific regularities of integrase interaction with DNA**

Fig. 11. SDS-PAGE analysis of products of [32P]casein hydrolysis by human milk sIgA and different proteases (A) or AIDS IgGs (B) (autoradiograph). A, Lanes 1, 2, and 3 correspond to [32P]-casein incubated with sIgAs for 20, 40, and 60 min, respectively; lanes 4 and 5, incubation with trypsin for 10 and 15 min; lanes 6 and 7, incubation with proteinase K for 10 and 15 min; lane 8, incubation with chymotrypsin for 10 min; 9, casein incubated alone for 60 min. The reaction mixture (10 l) for analysis of proteolytic activity of different proteases contained 6.2 g/ml casein and sIgAs or enzymes at the following concentrations: 0.32 g/ml trypsin, 0.064 g/ml chymotrypsin, 0.1 g/ml proteinase K, and 20 g/ml sIgA. B, [32P]casein was incubated for 14 h in the absence of Abs (lane 1) and in the presence IgGs from the sera of different AIDS patients: lane 2, IgG-1 (14 h), lanes 3 and 4, IgG2 (7 and 14 h, respectively), lane 5, IgG-3 (14 h).

Parenteral or oral administration of various proteins to animals late in pregnancy leads to the production of the corresponding Abs at high levels (Fey *et al.*, 1973; Mestecky *et al.*, 1987). Thus, pregnant women may be effectively immunized by contacts with compounds of viruses and bacteria that are not immunogenic in other healthy humans. There may be also some degree of autoimmunization during pregnancy similar to that in AI patients (Nevinsky et al., 2005, 2010a, 2010b and refs therein).

It was shown that lactation is associated with the appearance of sIgA and IgG abzymes with DNase, RNase (Kanyshkova *et al.*, 1997; Nevinsky *et al.*, 2000a, 2000b), ATPase (Semenov *et al.*, 2004), amylolytic (Savel'ev *et al.*, 2001), protein- (Nevinsky *et al.*, 1998), lipid- (Gorbunov *et al.*, 2005) and polysaccharide (Karataeva *et al.*, 2006a, 2006b) kinase activities in human milk. The specific activities of milk Abzs are significantly higher than those of Abzs from the blood of healthy lactating women and patients with different AI pathologies (Nevinsky *et al.,* 2003, 2005, 2010a, 2010b and refs therein).

We have compared the hydrolysis of -casein by canonical proteases, human milk sIgA (Fig. 11A) and three different individual AIDS IgGs (Fig. 11B) (Odintsova et al., 2006b; 2011). The patterns of -casein hydrolysis by milk sIgA, AIDS IgG, trypsin, chymotrypsin, and proteinase K were quite different. In addition, there was observed remarkable difference in the hydrolysis of -casein by three individual AIDS IgG-1, IgG-2, and IgG-3 (Fig 11B).

It should be mentioned that AIDS IgGs demonstrated only serine-like protease activity (Odintsova et al., 2006b), while milk IgAs additionally possess Me-dependent activity (Odintsova et al., 2011). Thus, possible ways of the production of Abzs with caseinhydrolyzing activity in healthy human mothers and in AIDS patients may be different.

## **6.2 Integrase hydrolysis by AIDS abzymes**

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

**94 67 43**

**B**

**1 2 3 4 5 kDa**

**67 43**

**30**

**20**

**14 2.**

**30**

**20**

**14 2.**

Fig. 11. SDS-PAGE analysis of products of [32P]casein hydrolysis by human milk sIgA and different proteases (A) or AIDS IgGs (B) (autoradiograph). A, Lanes 1, 2, and 3 correspond to [32P]-casein incubated with sIgAs for 20, 40, and 60 min, respectively; lanes 4 and 5, incubation with trypsin for 10 and 15 min; lanes 6 and 7, incubation with proteinase K for 10 and 15 min; lane 8, incubation with chymotrypsin for 10 min; 9, casein incubated alone for 60 min. The reaction mixture (10 l) for analysis of proteolytic activity of different proteases contained 6.2 g/ml casein and sIgAs or enzymes at the following concentrations: 0.32 g/ml trypsin, 0.064 g/ml chymotrypsin, 0.1 g/ml proteinase K, and 20 g/ml sIgA. B, [32P]casein was incubated for 14 h in the absence of Abs (lane 1) and in the presence IgGs from the sera of different AIDS patients: lane 2, IgG-1 (14 h), lanes 3 and 4, IgG2 (7 and 14 h,

Parenteral or oral administration of various proteins to animals late in pregnancy leads to the production of the corresponding Abs at high levels (Fey *et al.*, 1973; Mestecky *et al.*, 1987). Thus, pregnant women may be effectively immunized by contacts with compounds of viruses and bacteria that are not immunogenic in other healthy humans. There may be also some degree of autoimmunization during pregnancy similar to that in AI patients

It was shown that lactation is associated with the appearance of sIgA and IgG abzymes with DNase, RNase (Kanyshkova *et al.*, 1997; Nevinsky *et al.*, 2000a, 2000b), ATPase (Semenov *et al.*, 2004), amylolytic (Savel'ev *et al.*, 2001), protein- (Nevinsky *et al.*, 1998), lipid- (Gorbunov *et al.*, 2005) and polysaccharide (Karataeva *et al.*, 2006a, 2006b) kinase activities in human milk. The specific activities of milk Abzs are significantly higher than those of Abzs from the blood of healthy lactating women and patients with different AI pathologies (Nevinsky *et*

We have compared the hydrolysis of -casein by canonical proteases, human milk sIgA (Fig. 11A) and three different individual AIDS IgGs (Fig. 11B) (Odintsova et al., 2006b; 2011). The patterns of -casein hydrolysis by milk sIgA, AIDS IgG, trypsin, chymotrypsin, and proteinase K were quite different. In addition, there was observed remarkable difference in the hydrolysis of -casein by three individual AIDS IgG-1, IgG-2, and IgG-3 (Fig 11B). It should be mentioned that AIDS IgGs demonstrated only serine-like protease activity (Odintsova et al., 2006b), while milk IgAs additionally possess Me-dependent activity (Odintsova et al., 2011). Thus, possible ways of the production of Abzs with caseinhydrolyzing activity in healthy human mothers and in AIDS patients may be different.

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

**A**

respectively), lane 5, IgG-3 (14 h).

(Nevinsky et al., 2005, 2010a, 2010b and refs therein).

*al.,* 2003, 2005, 2010a, 2010b and refs therein).

## **6.2.1 Specific regularities of integrase interaction with DNA**

HIV-1 integrase catalyzes insertion of a DNA copy of the viral genome into the host genome (Asante-Appiah & Skalka, 1999)*.* Therefore IN, together with RT and protease, is the main important target of anti-HIV drugs.

Specific interactions between HIV IN and long terminal repeats are required for insertion of viral DNA into the host genome. The use of a method based on stepwise increase in ligand complexity allowed an estimation of the relative contributions of each nucleotide from oligonucleotides (ODNs) to the total affinity for IN (Bugreev et al., 2003). It was shown that IN interacts with ODNs through superposition of weak contacts with their bases and, more importantly, with the internucleotide phosphate groups. Formation of the IN complex with specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the *kcat* term; the rate of 3'-processin reaction is increased by more than 5 orders of magnitude upon transition from non-specific to specific oligonucleotides (Bugreev et al., 2003).

In solution, HIV-1 IN exists in a dynamic equilibrium of monomers, dimers, tetramers and high-order oligomers (Deprez et al., 2000). We have recently analyzed the activity of different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking and shown that these forms do not share the same *in vitro* catalytic properties (Faure et al., 2005). While monomers were inactive for all specific IN activities, dimers were able to catalyze the 3'-processing and the insertion of only one LTR into a short target DNA. In contrast, a tetramer of IN catalyzed the full-site integration of the two viral LTR ends into a target DNA.

To characterize the influence of the determinants of DNA substrate specificity on the oligomerization status of IN, the small-angle X-ray scattering technique was used (Baranova et al., 2007). Under special conditions in the absence of ODNs IN existed only as monomers. IN preincubation with specific ODNs led mainly to formation of dimers, the relative amount of which correlated well with the increase in the enzyme activity. Under these conditions, tetramers were scarce. Nonspecific ODNs stimulated formation of catalytically inactive dimers and tetramers. Complexes of monomeric, dimeric and tetrameric forms of IN with specific and nonspecific ODNs had varying radii of gyration (Rg), suggesting that the specific sequence-dependent formation of IN tetramers occurs by dimerization of two dimers of different structure. From the data it was concluded that the DNA-induced oligomerization of HIV-1 IN is of extreme importance to provide substrate specificity and to increase the enzyme activity (Baranova et al., 2007).

#### **6.2.2 Effect of DNA on the integrase hydrolysis by different proteases**

It is known that a formation of multiple contacts between the same or various subunits of oligomeric enzymes is usually provided by multiple hydrophobic and electrostatic contacts and hydrogen bonds. A similar situation was observed for the dimeric forms of HIV-1 IN by X-ray crystallography (for review see Wlodawer 1999; Chiu & Davies, 2004 ). Analysis of effects of specific and nonspecific ODNs on the rate of IN proteolysis by chymotrypsin, trypsin, and proteinase K can provide useful information concerning a possible decrease in the accessibility of aromatic and positively charged amino acid residues after an enzyme binds its substrates, changes its conformation, or forms contacts between its subunits. It was interesting to compare the effect of different ODNs on the cleavage of IN by Abzs and

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 179

at the carboxyl side of lysine and arginine residues (Fig. 12). Thus, these ODNs most probably stimulate formation of dimeric forms of IN with more Lys- and Arg-dependent electrostatic contacts between the monomers. A weak protective effect of specific and nonspecific ODNs was observed in the case of proteinase K, which is mostly sequenceindependent. Thus, specific and nonspecific DNAs stimulate the formation of different IN oligomeric forms, in which aromatic and charged amino acid residues in different extent accessible for Abzs, chymotrypsin, and trypsin. The findings correlate with the results obtained by small-angle X-ray scattering, which show that all nonspecific and specific ODNs stimulate different changes in the structure of IN monomers and dimers free of DNA

MALDI-TOF analysis of the fragments formed after IN incubation with pIgG and pIgM purified on IN-Sepharose was carried out (Odintsova E., Baranova S., and Nevinsky G.A., personal communication). The cross-sections of longitudinal slices of the gel corresponding to the products with approximate mol. masses 29 ± 2 (P1), 22 ± 2 (P2), 16 ± 2 (P3), and 12 ± 2 (P4), as well as 30± 2 (P0) kDa were cut out as shown on Fig. 13 and the proteins were eluted from the gel. MALDI-TOF analysis has shown that P1–P4 fractions contain from four to eight major fragments with different molecular masses. The peptides found in the P1–P4 fractions were digested with trypsin under standard conditions for MALDI analysis, and the

> P1 P2 P3 P4

Fig. 13. SDS-PAGE analysis of IN hydrolysis by pIgG-1 (lane 3, A), IgG-6 (lane 2, B), pIgM-2 (lane 4, A) and pIgM-6 (lane 3, B) purified on IN-Sepharose after 9 h (A) and 2 h (B) of incubation in a nonreducing 12% gel followed by silver staining. Lanes 1 (B) and 2 (A), IN incubated in the absence of Abs. Lane 1 (A) protein molecular mass markers. Gel zones P1, P2, P3, and P4 (A) as well as P0 (B) were used for MALDI-TOF analysis (see the text).

Seven antigenic determinants (AGDs) have been reported for HIV IN corresponding to amino acid residues 5–22 (AGD1), 14–35 (AGD2) (Yi et al., 2000), 58–141 (AGD3), 141–172 (AGD4), 248–264 (AGD5) (Bizub-Bender et al., 1994), 208–228 (AGD6), and 251–271(AGD7) (Nilsen et al., 1996) (underlined in Fig. 14). Interestingly, 6–7 cleavage sites found by MALDI corresponded to the N-terminal stretch of residues 11–35, belonging to two overlapping antigenic determinants AGD1 and AGD2 (Fig. 14). Three clusters of cleavage sites were located within the long AGD3. A block of 12 closely spaced cleavage sites corresponded to the N-terminal part of AGD4. Only one cleavage site was located within AGD5 and four sites corresponded to AGD6 and AGD7. At the same time, some sites of IN cleavage, most notably a cluster of 16 sites between residues 175 and 202, did not corresponds to any IN

P0

**1 2 3 4**

**A**

P0

**1 2 3 B**

P1

P0

P2 P3 P4

(Baranova et al., 2007).

hydrolyzates were studied.

AGD known at this moment.

**14.4 18.4 25.0**

**35.0 kDa**

canonical proteases. The specific single-stranded (ss) 5'-GTGTGGAAAATCTCTAGCA (19- CA), ss 5'-GTGTGGAAAATCTCTAGCAGT (21-GT), ss 5'-ACTGCTAGAGATTTTCCACAC (21-COM, complementary to 21-GT and to 19-CA), double-stranded (ds) 21-GT (21-GT•21- COM) and ds 19-CA (19-CA•21-COM) corresponding to terminal repeats of viral DNA were used.

While nonspecific d(pT)n markedly decreased the IgG-dependent hydrolysis of IN, d(pA)n and d(pA)n•d(pT)n demonstrated no detectable protective effect (Fig. 12) (Odintsova E., Baranova S., and Nevinsky G.A., personal communication).

Fig. 12. SDS-PAGE analysis of HIV IN hydrolysis by IgGs and canonical proteases. Polyclonal IgGs from HIV-infected patients (A), chymotrypsin (B), trypsin (C), and proteinase K (D) were used for hydrolysis of IN after its preincubation in the absence and in the presence of various ODNs leading to the formation of different IN oligomeric forms. IN was pre-incubated for 1 h at 30°C in the absence (lane 7) or in the presence of saturating concentrations (0.2–1 mM) of ss d(pA)24 (lane 1), ss d(pT)24 (lane 2), ds d(pA)24•d(pT)24 (lane 3), ss 21-COM (lane 4), ss 21-GT (lane 5), ds 21-GT•21-COM (lane 6). Then, preincubated mixture was diluted 3-fold and one of three canonical proteases or pIgGs from HIV-infected patients was added. After 5–10 min incubation in the presence of trypsin (19 M), chymotrypsin (1.6 M), proteinase K (19 M) and 6 h in the presence of pIgGs (0.17 M) the reaction was stopped and the efficiency of IN hydrolysis was analysed by SDS-PAGE.

The best protection from the hydrolysis by IgGs was observed for ss and especially ds specific ODNs (Fig. 12). Overall, the protective effects of all specific and nonspecific ss and ds ODNs from hydrolysis of IN by chymotrypsin were comparable. Therefore, one can suggest that the formation of IN complex with specific and nonspecific ODNs led to a similar decrease in the accessibility of aromatic amino acid residues as a result of their shielding by ODNs and/or involvement of these residues to the formation of multiple contacts at the interfaces of IN oligomer subunits. In contrast to chymotrypsin, nonspecific ODNs strongly protects IN from hydrolysis by trypsin, which cleaves peptide chains mainly

canonical proteases. The specific single-stranded (ss) 5'-GTGTGGAAAATCTCTAGCA (19- CA), ss 5'-GTGTGGAAAATCTCTAGCAGT (21-GT), ss 5'-ACTGCTAGAGATTTTCCACAC (21-COM, complementary to 21-GT and to 19-CA), double-stranded (ds) 21-GT (21-GT•21- COM) and ds 19-CA (19-CA•21-COM) corresponding to terminal repeats of viral DNA

While nonspecific d(pT)n markedly decreased the IgG-dependent hydrolysis of IN, d(pA)n and d(pA)n•d(pT)n demonstrated no detectable protective effect (Fig. 12) (Odintsova E.,

**kDa**

**14.4 18.4 25.0 35.0**

**kDa**

**14.4 18.4 25.0 35.0**

**1 2 3 4 5 6 7 C M 1 2 3 4 5 6 7 C M**

 **1 2 3 4 5 6 7 C M 1 2 3 4 5 6 7 C M**

Fig. 12. SDS-PAGE analysis of HIV IN hydrolysis by IgGs and canonical proteases. Polyclonal IgGs from HIV-infected patients (A), chymotrypsin (B), trypsin (C), and

proteinase K (D) were used for hydrolysis of IN after its preincubation in the absence and in the presence of various ODNs leading to the formation of different IN oligomeric forms. IN was pre-incubated for 1 h at 30°C in the absence (lane 7) or in the presence of saturating concentrations (0.2–1 mM) of ss d(pA)24 (lane 1), ss d(pT)24 (lane 2), ds d(pA)24•d(pT)24 (lane 3), ss 21-COM (lane 4), ss 21-GT (lane 5), ds 21-GT•21-COM (lane 6). Then, preincubated mixture was diluted 3-fold and one of three canonical proteases or pIgGs from HIV-infected patients was added. After 5–10 min incubation in the presence of trypsin (19 M), chymotrypsin (1.6 M), proteinase K (19 M) and 6 h in the presence of pIgGs (0.17 M) the reaction was stopped and the efficiency of IN hydrolysis was analysed by

The best protection from the hydrolysis by IgGs was observed for ss and especially ds specific ODNs (Fig. 12). Overall, the protective effects of all specific and nonspecific ss and ds ODNs from hydrolysis of IN by chymotrypsin were comparable. Therefore, one can suggest that the formation of IN complex with specific and nonspecific ODNs led to a similar decrease in the accessibility of aromatic amino acid residues as a result of their shielding by ODNs and/or involvement of these residues to the formation of multiple contacts at the interfaces of IN oligomer subunits. In contrast to chymotrypsin, nonspecific ODNs strongly protects IN from hydrolysis by trypsin, which cleaves peptide chains mainly

Baranova S., and Nevinsky G.A., personal communication).

**A B**

**C D**

were used.

SDS-PAGE.

at the carboxyl side of lysine and arginine residues (Fig. 12). Thus, these ODNs most probably stimulate formation of dimeric forms of IN with more Lys- and Arg-dependent electrostatic contacts between the monomers. A weak protective effect of specific and nonspecific ODNs was observed in the case of proteinase K, which is mostly sequenceindependent. Thus, specific and nonspecific DNAs stimulate the formation of different IN oligomeric forms, in which aromatic and charged amino acid residues in different extent accessible for Abzs, chymotrypsin, and trypsin. The findings correlate with the results obtained by small-angle X-ray scattering, which show that all nonspecific and specific ODNs stimulate different changes in the structure of IN monomers and dimers free of DNA (Baranova et al., 2007).

MALDI-TOF analysis of the fragments formed after IN incubation with pIgG and pIgM purified on IN-Sepharose was carried out (Odintsova E., Baranova S., and Nevinsky G.A., personal communication). The cross-sections of longitudinal slices of the gel corresponding to the products with approximate mol. masses 29 ± 2 (P1), 22 ± 2 (P2), 16 ± 2 (P3), and 12 ± 2 (P4), as well as 30± 2 (P0) kDa were cut out as shown on Fig. 13 and the proteins were eluted from the gel. MALDI-TOF analysis has shown that P1–P4 fractions contain from four to eight major fragments with different molecular masses. The peptides found in the P1–P4 fractions were digested with trypsin under standard conditions for MALDI analysis, and the hydrolyzates were studied.

Fig. 13. SDS-PAGE analysis of IN hydrolysis by pIgG-1 (lane 3, A), IgG-6 (lane 2, B), pIgM-2 (lane 4, A) and pIgM-6 (lane 3, B) purified on IN-Sepharose after 9 h (A) and 2 h (B) of incubation in a nonreducing 12% gel followed by silver staining. Lanes 1 (B) and 2 (A), IN incubated in the absence of Abs. Lane 1 (A) protein molecular mass markers. Gel zones P1, P2, P3, and P4 (A) as well as P0 (B) were used for MALDI-TOF analysis (see the text).

Seven antigenic determinants (AGDs) have been reported for HIV IN corresponding to amino acid residues 5–22 (AGD1), 14–35 (AGD2) (Yi et al., 2000), 58–141 (AGD3), 141–172 (AGD4), 248–264 (AGD5) (Bizub-Bender et al., 1994), 208–228 (AGD6), and 251–271(AGD7) (Nilsen et al., 1996) (underlined in Fig. 14). Interestingly, 6–7 cleavage sites found by MALDI corresponded to the N-terminal stretch of residues 11–35, belonging to two overlapping antigenic determinants AGD1 and AGD2 (Fig. 14). Three clusters of cleavage sites were located within the long AGD3. A block of 12 closely spaced cleavage sites corresponded to the N-terminal part of AGD4. Only one cleavage site was located within AGD5 and four sites corresponded to AGD6 and AGD7. At the same time, some sites of IN cleavage, most notably a cluster of 16 sites between residues 175 and 202, did not corresponds to any IN AGD known at this moment.

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 181

substrates with both light and heavy chains of Abzs. The separation of the light chains can lead to a decrease in the lifetime of the existence of the complex and, as a consequence, to an increase in the turnover number and *Vmax* (*kca*t) of the reaction catalyzed by L-chains*.* Taken together, the absence or very weak interaction of short substrates with heavy chains of AIDS Abzs in contrast with globular molecules proteins (and higher rate of the reaction) may be a main reason of a decrease of specificity of Abzs action in the case of short oligopeptides; one cannot exclude that light (or heavy) chains of some Abzs can effectively hydrolyzed short

We have shown that *in vitro* IgGs and IgMs hydrolyzing IN significantly decrease the 3' processing and integration reaction catalyzed by IN (for example, Fig. 15) (Odintsova E.,

**[IgG], M**

**01234567**

**2.8 M**

**0.05 0.10 0.15 0.20**

**[IgM], M**

According to modern point of view, the immune response to the viral components is the most important factor providing slow transition of HIV infection to the stage of AIDS (Fauci et al., 2008). Since AIDS anti-IN anti-RT Abs can efficiently hydrolyze IN and RT (Odintsova et al., 2006b; Baranova et al., 2009, 2010), a positive role of abzymes in counteracting the infection cannot be excluded and these polyclonal and corresponding monoclonal Abzs with proteolytic activities are potentially interesting for designing new anti-HIV agents. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for

The field of monoclonal Abzs with immunotherapeutic potential has recently been reviewed (see "Introduction"). Abzs that cleave HIV envelope gp120 protein may find their use in the treatment of AIDS (Tellier, 2002; Stockwin & Holmes, 2003). pIgG degrading gp120 was also obtained taking advantage of the susceptibility of SJL mice to a peptide-induced AI disorder, experimental AI encephalomyelitis (Ponomarenko et al., 2006). Immunization of specific pathogen-free SJL mice with structural fragments of gp120 fused in-frame with the encephalitogenic MBP(85-101) peptide resulted in a pronounced disease-associated immune response against these antigens. This strategy can be generalized to create catalytic vaccines against viral pathogens (Ponomarenko et al., 2006). In addition, Abzs with different catalytic

Fig. 15. Effect of IN-hydrolyzing IgGs and IgMs from AIDS patients on the rate of the 3'-

oligopeptides of any sequences.

processing reaction.

**7. Conclusion**

Baranova S., and Nevinsky G.A., personal communication).

**Relative activity, %**

assessment of the immune status in AIDS patients.

activities can be used for different purposes (see "Introduction").

**0.05 M**

Fig. 14. All trypsin-independent points of IN cleavage determined from the MALDI-TOF analysis directly mol. masses of the P0–P4 fractions and after their cleavage by trypsin. Numbers next to the arrows show the position of the residue within the IN sequence; numbers under short arrows and in parentheses correspond to the frequency of these cleavage sites found using different approaches. Seven known antigenic determinants of IN are underlined in the figure.

Interestingly, a similar situation was observed for MS IgGs specifically hydrolyzing MBP; in addition to the sites of cleavage within four known AGDs of MBP, several sites were outside these determinants (Ponomarenko et al., 2006). Thus, the number of structurally different antigenic determinants in the case of IN may be great.

Interestingly, in contrast to the absence of hydrolysis of non-specific globular proteins by anti-IN Abs, they first cleave IN with the accumulation of long fragments corresponding mainly to known AGDs and then are capable of further degradation of these long intermediates, and the formation of very short products was observed after 72–100 h of IN incubation In with Abzs (Odintsova E., Baranova S., and Nevinsky G.A., personal communication). AIDS anti-IN pIgGs and IgMs hydrolyze specific 20-25-mer oligopeptides corresponding to the IN AGDs ~30–70-fold faster than nonspecific long 20-25-mer oligopeptides corresponding to AGDs of human myelin basic protein and HIV RT. In addition, AIDS anti-IN Abzs can hydrolyze very short 3-4-mer nonspecific oligopeptides 100–300-fold more slowly than specific ones. Therefore, the recognition and digestion of globular proteins and relatively short oligopeptides by the Abzs proceeds in different ways. Since catalytic centers of Abzs specifically hydrolyzing different proteins including IN are usually located on the light chains of Abs (Nevinsky et al., 2005, 2010a, 2010b, and refs therein), the observed hydrolysis of short oligopeptides can be a consequence of their interaction with light or heavy chains without significant contacts with alternative chains.

Interestingly, separated light chains of pIgGs, pIgMs, and pIgAs from the sera of patients with different AI and viral diseases usually significantly more active than intact Abs in the hydrolysis of DNA, RNA, oligosaccharides, and proteins (Nevinsky et al., 2005, 2010a, 2010b, and refs therein). This phenomenon may be a consequence of a higher affinity of intact Abs, as compared with separated light chains, for different substrates due to interaction of the substrates with both light and heavy chains of Abzs. The separation of the light chains can lead to a decrease in the lifetime of the existence of the complex and, as a consequence, to an increase in the turnover number and *Vmax* (*kca*t) of the reaction catalyzed by L-chains*.* Taken together, the absence or very weak interaction of short substrates with heavy chains of AIDS Abzs in contrast with globular molecules proteins (and higher rate of the reaction) may be a main reason of a decrease of specificity of Abzs action in the case of short oligopeptides; one cannot exclude that light (or heavy) chains of some Abzs can effectively hydrolyzed short oligopeptides of any sequences.

We have shown that *in vitro* IgGs and IgMs hydrolyzing IN significantly decrease the 3' processing and integration reaction catalyzed by IN (for example, Fig. 15) (Odintsova E., Baranova S., and Nevinsky G.A., personal communication).

Fig. 15. Effect of IN-hydrolyzing IgGs and IgMs from AIDS patients on the rate of the 3' processing reaction.

## **7. Conclusion**

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

MALNGIDKA E DHEKYHSN R AMVSDFNL PVVA K EIVA CDKCQL KGE MHGQVDC -57 **Q W PS A**

**90-91 (1) 92-93 (2)**

**E V YPS**

**145-156 (1)**

**157-158 (1)**

2 1 2 1 2 4 1 1 4 4 1

**179-180 (1)**

**A F G KA A**

**158-159 (1)**

4 4 5 1 5 1 4 3 3 2 3 2 13 1 3 1

KGE **PS**

**159-160 (1)**

**113-114 (1)**

**G RQ K D A**

**AGD5**

SP**G**IWQLDCTHL GKIILVAVH ASGYIE AEV I PA ETGQETA FLLKLAGRW VKT IHTDNG

**147-148 (1)**

**86-87 (1) 89-90 (1)**

**AGD1 AGD2**

2 2 2 1 3 3 1

**191-192 (1)**

**247-248 (2) 250-251 (2) 259-260 (2)**

3 1 1 3 3 1 1 2 2

**AGD6 AGD7**

**AGD3**

**120-121 (1) 127-128 (1)**

**184-185 (1) 189-190 (1)**

2 2

are underlined in the figure.

**11-12 (1) 21-22 (4) 34-35 (1) 35-36 (2) 46-47 (1)**

NFTSTTV KA CWWAGIKQE GIPYNPQ S Q VVESM NN E L KIIGQVRDQ EHLKTAVQM

**148-149 (1)**

Fig. 14. All trypsin-independent points of IN cleavage determined from the MALDI-TOF analysis directly mol. masses of the P0–P4 fractions and after their cleavage by trypsin. Numbers next to the arrows show the position of the residue within the IN sequence; numbers under short arrows and in parentheses correspond to the frequency of these cleavage sites found using different approaches. Seven known antigenic determinants of IN

Interestingly, a similar situation was observed for MS IgGs specifically hydrolyzing MBP; in addition to the sites of cleavage within four known AGDs of MBP, several sites were outside these determinants (Ponomarenko et al., 2006). Thus, the number of structurally different

Interestingly, in contrast to the absence of hydrolysis of non-specific globular proteins by anti-IN Abs, they first cleave IN with the accumulation of long fragments corresponding mainly to known AGDs and then are capable of further degradation of these long intermediates, and the formation of very short products was observed after 72–100 h of IN incubation In with Abzs (Odintsova E., Baranova S., and Nevinsky G.A., personal communication). AIDS anti-IN pIgGs and IgMs hydrolyze specific 20-25-mer oligopeptides corresponding to the IN AGDs ~30–70-fold faster than nonspecific long 20-25-mer oligopeptides corresponding to AGDs of human myelin basic protein and HIV RT. In addition, AIDS anti-IN Abzs can hydrolyze very short 3-4-mer nonspecific oligopeptides 100–300-fold more slowly than specific ones. Therefore, the recognition and digestion of globular proteins and relatively short oligopeptides by the Abzs proceeds in different ways. Since catalytic centers of Abzs specifically hydrolyzing different proteins including IN are usually located on the light chains of Abs (Nevinsky et al., 2005, 2010a, 2010b, and refs therein), the observed hydrolysis of short oligopeptides can be a consequence of their interaction with light or heavy chains without significant contacts with alternative chains. Interestingly, separated light chains of pIgGs, pIgMs, and pIgAs from the sera of patients with different AI and viral diseases usually significantly more active than intact Abs in the hydrolysis of DNA, RNA, oligosaccharides, and proteins (Nevinsky et al., 2005, 2010a, 2010b, and refs therein). This phenomenon may be a consequence of a higher affinity of intact Abs, as compared with separated light chains, for different substrates due to interaction of the

VFIH NFKRK G IGGYSAGE IVDIIATDI T KELQKQIT IQNFRVYYR NKDPLWKGP

**211-212 (1)**

KLLWKGE GA VIQDNSDIK VPRRKAKII DYGKQMAGD CVASRQDED

**AGD4**

2 1

**V VR D**

**139-140 (2)**

1

antigenic determinants in the case of IN may be great.

According to modern point of view, the immune response to the viral components is the most important factor providing slow transition of HIV infection to the stage of AIDS (Fauci et al., 2008). Since AIDS anti-IN anti-RT Abs can efficiently hydrolyze IN and RT (Odintsova et al., 2006b; Baranova et al., 2009, 2010), a positive role of abzymes in counteracting the infection cannot be excluded and these polyclonal and corresponding monoclonal Abzs with proteolytic activities are potentially interesting for designing new anti-HIV agents. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for assessment of the immune status in AIDS patients.

The field of monoclonal Abzs with immunotherapeutic potential has recently been reviewed (see "Introduction"). Abzs that cleave HIV envelope gp120 protein may find their use in the treatment of AIDS (Tellier, 2002; Stockwin & Holmes, 2003). pIgG degrading gp120 was also obtained taking advantage of the susceptibility of SJL mice to a peptide-induced AI disorder, experimental AI encephalomyelitis (Ponomarenko et al., 2006). Immunization of specific pathogen-free SJL mice with structural fragments of gp120 fused in-frame with the encephalitogenic MBP(85-101) peptide resulted in a pronounced disease-associated immune response against these antigens. This strategy can be generalized to create catalytic vaccines against viral pathogens (Ponomarenko et al., 2006). In addition, Abzs with different catalytic activities can be used for different purposes (see "Introduction").

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients 183

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In conclusion, a number of studies of Abzs show the extremely wide potential of the immune system in producing Abzs possessing very different enzymatic activities, which very often are not comparable with those of known enzymes, and natural Abs with specified and novel functions may have wide potential for biotechnology and medicine.

## **8. Acknowledgments**

This research was made possible in part by grants from Analytical Departmental Special program "Development of Scientific Potential of High School (No. 2.1.1/5580)" from the Presidium of the Russian Academy of Sciences ("Molecular and Cellular Biology Program" No. 22.7, "Fundamental Sciences to Medicine" No. 21.16), Russian Foundation for Basic Research (08-04-00315, 09-04-00804), and funds from the Siberian Division of the Russian Academy of Sciences.

## **9. Abbreviations**

Abs - antibodies Abzs - abzymes - or catalytically active antibodies AG - antigen AI - autoimmune AD - autoimmune disease AIDS - human autoimmune deficit syndrome BSA - bovine serum albumin CC - correlation coefficient HSCs - hematopoietic stem cells sc - supercoiled ss and ds - single- and double-stranded - respectively CBA - (CBAxC57BL)F1 mice HT - Hashimoto's thyroiditis hMBP - human myelin basic protein MFT - microsomal fraction of thyrocytes MBP - myelin basic protein MS - multiple sclerosis nat-DNA and den-DNA - native and denatured DNA - respectively MHO - maltoheptaose pAbs and pIgGs - polyclonal Abs and IgGs - respectively RF - rheumatoid factor SLE- systemic lupus erythematosus - SDS-PAGE - SDS-polyacrylamide gel electrophoresis TBE - tick-borne encephalitis VIP - vasoactive intestinal peptide RA - relative activity CFU-GM - granulocytic-macrophagic colony-forming unit BFU-E - erythroid burst-forming unit CFU-GEMM - granulocytic-erythroid-megakaryocytic- macrophagic colony-forming unit

#### **10. References**

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

In conclusion, a number of studies of Abzs show the extremely wide potential of the immune system in producing Abzs possessing very different enzymatic activities, which very often are not comparable with those of known enzymes, and natural Abs with specified

This research was made possible in part by grants from Analytical Departmental Special program "Development of Scientific Potential of High School (No. 2.1.1/5580)" from the Presidium of the Russian Academy of Sciences ("Molecular and Cellular Biology Program" No. 22.7, "Fundamental Sciences to Medicine" No. 21.16), Russian Foundation for Basic Research (08-04-00315, 09-04-00804), and funds from the Siberian Division of the Russian

and novel functions may have wide potential for biotechnology and medicine.

**8. Acknowledgments**

Academy of Sciences.

**9. Abbreviations**  Abs - antibodies

AD - autoimmune disease

BSA - bovine serum albumin CC - correlation coefficient HSCs - hematopoietic stem cells

CBA - (CBAxC57BL)F1 mice HT - Hashimoto's thyroiditis

MBP - myelin basic protein MS - multiple sclerosis

MHO - maltoheptaose

RF - rheumatoid factor

RA - relative activity

TBE - tick-borne encephalitis VIP - vasoactive intestinal peptide

BFU-E - erythroid burst-forming unit

hMBP - human myelin basic protein MFT - microsomal fraction of thyrocytes

AG - antigen AI - autoimmune

sc - supercoiled

Abzs - abzymes - or catalytically active antibodies

AIDS - human autoimmune deficit syndrome

ss and ds - single- and double-stranded - respectively

nat-DNA and den-DNA - native and denatured DNA - respectively

SLE- systemic lupus erythematosus - SDS-PAGE - SDS-polyacrylamide gel electrophoresis

CFU-GEMM - granulocytic-erythroid-megakaryocytic- macrophagic colony-forming unit

pAbs and pIgGs - polyclonal Abs and IgGs - respectively

CFU-GM - granulocytic-macrophagic colony-forming unit

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**10**

*South Africa*

**RNAi-Based Gene Expression**

Fiona T. van den Berg and Marc S. Weinberg

*Antiviral Gene Therapy Research Unit (AGTRU), University of the Witwatersrand*

Antiretroviral drugs have made a significant impact on HIV/AIDS disease progression and have significantly extended the life expectancy of HIV-infected individuals, particularly when used in combination therapies such as HAART (highly active antiretroviral therapy). However, despite this success, recent reports indicate that HIV morbidity and mortality remain at epidemic proportions. It is estimated that over 33 million people are currently infected with the virus worldwide, while sub-Saharan Africa accounted for approximately 70% of all infected individuals and AIDS-related deaths in 2009 (UNAIDS, 2010). Issues of drug resistance, drug toxicity, correct patient compliance and the inability to remove latent reservoirs of infection remain significant problems to overcome. The need to develop novel and improved therapeutic strategies against HIV therefore remains an important medical objective. Gene-based therapies hold much promise as alternative treatment strategies for HIV/AIDS. Anti-viral gene therapies aim to provide a once-off, long-lasting treatment against the virus and thereby address some of the shortcomings associated with current

A gene therapy against HIV offers several unique advantages, including the sustained inhibition of viral replication and the removal of virus from cellular reservoirs. Moreover, by improving specificity, the common toxic side effects associated with current antiretrovial regimens can be diminished. A number of different RNA-based and protein-based gene therapy strategies have been explored and some have reached phase 1 and 2 clinical trials. Our research focuses on the development of RNA-based antiviral strategies and in particular, those that utilise gene expression strategies based on RNA interference (RNAi). In this chapter, we examine basic concepts and review recent advances in the development of expressed RNAibased systems against HIV, with a focus on progress in construct and target design. We also discuss topics related to the use of RNAi-based strategies, including appropriate construct expression, target specificity, viral escape mutations and effective construct delivery. We aim to identify desirable properties of an RNAi-based anti-HIV therapy and highlight the future

RNA interference (RNAi) is a gene silencing phenomenon in which RNA molecules act to silence the expression of particular genes at a post-transcriptional level in the cell. RNAi has

developments that are required to make this approach a reality.

**1. Introduction**

antiretroviral therapies.

**2. RNA interference**

**Strategies to Combat HIV**

## **RNAi-Based Gene Expression Strategies to Combat HIV**

Fiona T. van den Berg and Marc S. Weinberg *Antiviral Gene Therapy Research Unit (AGTRU), University of the Witwatersrand South Africa*

## **1. Introduction**

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Antiretroviral drugs have made a significant impact on HIV/AIDS disease progression and have significantly extended the life expectancy of HIV-infected individuals, particularly when used in combination therapies such as HAART (highly active antiretroviral therapy). However, despite this success, recent reports indicate that HIV morbidity and mortality remain at epidemic proportions. It is estimated that over 33 million people are currently infected with the virus worldwide, while sub-Saharan Africa accounted for approximately 70% of all infected individuals and AIDS-related deaths in 2009 (UNAIDS, 2010). Issues of drug resistance, drug toxicity, correct patient compliance and the inability to remove latent reservoirs of infection remain significant problems to overcome. The need to develop novel and improved therapeutic strategies against HIV therefore remains an important medical objective. Gene-based therapies hold much promise as alternative treatment strategies for HIV/AIDS. Anti-viral gene therapies aim to provide a once-off, long-lasting treatment against the virus and thereby address some of the shortcomings associated with current antiretroviral therapies.

A gene therapy against HIV offers several unique advantages, including the sustained inhibition of viral replication and the removal of virus from cellular reservoirs. Moreover, by improving specificity, the common toxic side effects associated with current antiretrovial regimens can be diminished. A number of different RNA-based and protein-based gene therapy strategies have been explored and some have reached phase 1 and 2 clinical trials. Our research focuses on the development of RNA-based antiviral strategies and in particular, those that utilise gene expression strategies based on RNA interference (RNAi). In this chapter, we examine basic concepts and review recent advances in the development of expressed RNAibased systems against HIV, with a focus on progress in construct and target design. We also discuss topics related to the use of RNAi-based strategies, including appropriate construct expression, target specificity, viral escape mutations and effective construct delivery. We aim to identify desirable properties of an RNAi-based anti-HIV therapy and highlight the future developments that are required to make this approach a reality.

## **2. RNA interference**

RNA interference (RNAi) is a gene silencing phenomenon in which RNA molecules act to silence the expression of particular genes at a post-transcriptional level in the cell. RNAi has

RNAi-Based Gene Expression Strategies to Combat HIV 195

Fig. 1. The mammalian microRNA biogenesis pathway which mediates RNAi. Artificial antiviral constructs can be introduced at points 1, 2 and 3 of the pathway and processed to

inhibit viral replication.

become a popular tool in the development of antiviral therapeutics as the potent silencing mechanism can be redirected against viral genes to inhibit viral replication. RNAi was first described by Fire *et. al*. (Fire *et al.*, 1998) and has since been demonstrated in a number of different organisms including yeast, plants and animals. The mediators of RNAi are short 21- and 22- nucleotide (nt) RNAs known as small interfering RNAs (siRNAs) or microRNAs (miRNAs) and are derived from longer double stranded (ds) RNAs. SiRNAs/miRNAs direct the silencing of complementary gene transcripts in a sequence-specific manner. RNAi was first described in mammalian cells in 2001 (Elbashir *et al.*, 2001a) and subsequent research has moved swiftly to reveal a number of pathway components and mechanisms. Mammalian RNAi is now emerging as a complex network with several alternative RNA forms and levels of regulatory interactions (Breving and Esquela-Kerscher, 2009); (Ding *et al.*, 2009). RNAi–based therapeutics, however, still make use of the central RNAi pathway involved in miRNA biogenesis (Figure 1). Our discussion of RNAi-based strategies begins here with a description of this major RNAi pathway and how it can be redirected to inhibit viral replication.

The mammalian RNAi pathway mediates gene silencing through the generation of miRNAs. MiRNA regulation of genes is both essential and ubiquitous and has been implicated in the regulation of developmental timing, cellular differentiation, apoptosis, cell proliferation and organ development (Bartel, 2004). MiRNAs are expressed from non-protein-coding genes in intergenic or intronic regions as single or polycistronic transcripts by RNA polymerase II (Lee *et al.*, 2004); (Cullen, 2004). MiRNA transcripts are usually several kilobases long and fold back upon themselves to form characteristic hairpin structures known as primarymicroRNAs (pri-miRNAs) with flanking sequences, a partially duplexed stem and a terminal loop. Pri-miRNAs are processed in the RNAi pathway in two successive enzymatic steps (Lee *et al.*, 2002b) to produce mature miRNAs from the double-stranded stem region.

The first processing step occurs in the nucleus where the pri-miRNA is cleaved asymmetrically by the "microprocessor" complex to produce a shorter ~ 70 nt hairpin known as a precursor-microRNA (pre-miRNA) with a 2 nt overhang at the 3' hydroxyl end. The microprocessor complex includes two essential proteins, namely, the RNAse III enzyme Drosha and the DGCR8 (DiGeorge critical region 8) protein (Han *et al.*, 2004). The premiRNA is exported from the nucleus to the cytoplasm by the nuclear karyopherin Exportin-5 (Exp-5) in a Ran-GTP-dependant manner (Yi *et al.*, 2003); (Lund *et al.*, 2004). In the second processing step, the pre-miRNA is cleaved asymmetrically by another RNase III enzyme, Dicer (Paddison *et al.*, 2002) to produce staggered ~22 base pair (bp) miRNA/miRNA\* duplex with 2 nt overhangs at each 3' hydroxyl end. Dicer is thought to form a complex with TRBP (TAR RNA-binding protein) (Chendrimada *et al.*, 2005) and PACT (protein activator of protein kinase PKR) (Lee *et al.*, 2006).

One strand of the miRNA/miRNA\* duplex is selected as the mature miRNA or guide strand and loaded into the RNA-induced silencing complex (RISC) (Martinez *et al.*, 2002a). The guide strand (miRNA) is typically selected from the duplex as a result of weaker 5' end pairing, while the remaining passenger strand (miRNA\*) is degraded (Khvorova *et al.*, 2003); (Schwarz *et al.*, 2003). In certain cases, both strands of the duplex may be capable of RISC incorporation. RISC facilitates sequence-specific gene silencing and is directed by the guide sequence to complementary regions in the 3' untranslated regions of target messenger RNAs. RISC-targeting results in the cleavage, degradation or translational suppression of a gene transcript, depending on the level of total complementation between the miRNA and

become a popular tool in the development of antiviral therapeutics as the potent silencing mechanism can be redirected against viral genes to inhibit viral replication. RNAi was first described by Fire *et. al*. (Fire *et al.*, 1998) and has since been demonstrated in a number of different organisms including yeast, plants and animals. The mediators of RNAi are short 21- and 22- nucleotide (nt) RNAs known as small interfering RNAs (siRNAs) or microRNAs (miRNAs) and are derived from longer double stranded (ds) RNAs. SiRNAs/miRNAs direct the silencing of complementary gene transcripts in a sequence-specific manner. RNAi was first described in mammalian cells in 2001 (Elbashir *et al.*, 2001a) and subsequent research has moved swiftly to reveal a number of pathway components and mechanisms. Mammalian RNAi is now emerging as a complex network with several alternative RNA forms and levels of regulatory interactions (Breving and Esquela-Kerscher, 2009); (Ding *et al.*, 2009). RNAi–based therapeutics, however, still make use of the central RNAi pathway involved in miRNA biogenesis (Figure 1). Our discussion of RNAi-based strategies begins here with a description of this major RNAi pathway and how it can be redirected to inhibit

The mammalian RNAi pathway mediates gene silencing through the generation of miRNAs. MiRNA regulation of genes is both essential and ubiquitous and has been implicated in the regulation of developmental timing, cellular differentiation, apoptosis, cell proliferation and organ development (Bartel, 2004). MiRNAs are expressed from non-protein-coding genes in intergenic or intronic regions as single or polycistronic transcripts by RNA polymerase II (Lee *et al.*, 2004); (Cullen, 2004). MiRNA transcripts are usually several kilobases long and fold back upon themselves to form characteristic hairpin structures known as primarymicroRNAs (pri-miRNAs) with flanking sequences, a partially duplexed stem and a terminal loop. Pri-miRNAs are processed in the RNAi pathway in two successive enzymatic steps (Lee *et al.*, 2002b) to produce mature miRNAs from the double-stranded stem region. The first processing step occurs in the nucleus where the pri-miRNA is cleaved asymmetrically by the "microprocessor" complex to produce a shorter ~ 70 nt hairpin known as a precursor-microRNA (pre-miRNA) with a 2 nt overhang at the 3' hydroxyl end. The microprocessor complex includes two essential proteins, namely, the RNAse III enzyme Drosha and the DGCR8 (DiGeorge critical region 8) protein (Han *et al.*, 2004). The premiRNA is exported from the nucleus to the cytoplasm by the nuclear karyopherin Exportin-5 (Exp-5) in a Ran-GTP-dependant manner (Yi *et al.*, 2003); (Lund *et al.*, 2004). In the second processing step, the pre-miRNA is cleaved asymmetrically by another RNase III enzyme, Dicer (Paddison *et al.*, 2002) to produce staggered ~22 base pair (bp) miRNA/miRNA\* duplex with 2 nt overhangs at each 3' hydroxyl end. Dicer is thought to form a complex with TRBP (TAR RNA-binding protein) (Chendrimada *et al.*, 2005) and PACT (protein activator

One strand of the miRNA/miRNA\* duplex is selected as the mature miRNA or guide strand and loaded into the RNA-induced silencing complex (RISC) (Martinez *et al.*, 2002a). The guide strand (miRNA) is typically selected from the duplex as a result of weaker 5' end pairing, while the remaining passenger strand (miRNA\*) is degraded (Khvorova *et al.*, 2003); (Schwarz *et al.*, 2003). In certain cases, both strands of the duplex may be capable of RISC incorporation. RISC facilitates sequence-specific gene silencing and is directed by the guide sequence to complementary regions in the 3' untranslated regions of target messenger RNAs. RISC-targeting results in the cleavage, degradation or translational suppression of a gene transcript, depending on the level of total complementation between the miRNA and

viral replication.

of protein kinase PKR) (Lee *et al.*, 2006).

Fig. 1. The mammalian microRNA biogenesis pathway which mediates RNAi. Artificial antiviral constructs can be introduced at points 1, 2 and 3 of the pathway and processed to inhibit viral replication.

RNAi-Based Gene Expression Strategies to Combat HIV 197

Fig. 2. Various forms of therapeutic constructs used to trigger antiviral RNAi. Each construct generates antiviral siRNA or miRNA guide sequences (green, yellow, red and blue) which initiate silencing of complementary viral targets. Constructs may be non-expressed or

expressed in a singular or multiple format (combinatorial-RNAi).