The escape of HIV-1 from effects of neutralizing antibodies includes mutations in gp120 and/or gp41. One of the seminal studies showed that A582T point mutation in the HIV-1 envelope gene made a laboratory adapted HIV-1 strain called HXB2 to be resistant to HIV-1 neutralizing human serum (Reitz *et al.*, 1988). Another study published ten years later, showed that HIV-1 MN molecular clones which had 294N/K mutation before the V3 loop, 307N/I mutation proximal to the V3 loop, 327I/K mutation within V3 and 336N/I mutation after the V3 loop were resistant to human serum containing neutralizing antibodies directed at the immunodominant V3 loop of gp120 (Park *et al.*, 1998). Point mutations in a highly conserved structural motif within the intracytoplasmic tail of gp41 rendered HIV-1 molecular clones resistant to broadly neutralizing polyclonal human serum antibodies (Kalia *et al.*, 2005).

Point Mutations Associated with HIV-1 Drug

Epitopes Example

Glycans 2G12

MPER of gp41

CD4 binding site(CD4bs) of mAb

IgGb12, VRC01, HJ16, PG9 and PG16

> 4E10, 2F5

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 111

perinatally infected child with F673L point mutation was resistant to 4E10 (Gray et al., 2008). These case studies are examples of the challenges presented by point mutations in controlling HIV-1 infection by antibodies. Notwithstanding, recent discovery of additional and more potent broadly neutralizing antibodies such as VRC01 (X. Wu *et al.*, 2010), PG9 and PG16 (Walker et al., 2009) is encouraging and boosts the impetus for HIV-1 vaccine

neutralization Properties Selected references

(Corti *et al.*, 2010; Euler *et al.*, 2011; Ho *et al.*, 1991; Y. Li *et al.*, 2011; Pancera *et al.*, 2010; Posner *et al.*, 1991; Walker *et al.*, 2009; X. Wu *et al.*, 2010; Zhou *et al.*, 2011)

(Binley *et al.*, 2006; Calarese *et al.*, 2003; Luo *et al.*, 2011; Scanlan *et al.*, 2003; Trkola *et al.*, 1996b)

(Muster *et al.*, 1994; Muster *et al.*, 1993; Ruprecht *et al.*, 2011; Zwick *et al.*, 2005; Zwick *et al.*, 2001)

CD4bs antibodies compete with CD4 and with antibodies against CD4i epitope. PG9 and PG16 bind to trimer specific glycosylated epitope on the V1/V2 and V3 region.

Antibody binding is dependent upon proper N-linked glycosylation. Recognize a mannose rich epitope on the "silent" face of gp120

Both antibodies bind few crucial residues in the MPER of gp41. HIV-1 2F5 binds both native and fusion-intermediate conformations while 4E10 recognize a predominantly linear and relatively conserved epitope.

Table 8. Broadly neutralizing antibodies that bind gp120 and gp41 HIV-1 envelope proteins

Aptamers are synthetic oligonucleotide ligands that can be isolated *in vitro* against diverse targets including HIV-1 gp120 (Khati *et al.*, 2003). Aptamers assume a defined threedimensional structure and generally bind functional sites on their respective targets. They possess the molecular recognition properties of monoclonal antibodies in terms of their high affinity and specificity (Khati, 2010). Unlike antibodies, aptamers can fold properly and retain activity in the intracellular environment. However, the majority of aptamers with potential therapeutic utility selected to date target extracellular proteins (Chen *et al.*, 2003; Eyetech Study Group, 2002, 2003; Green *et al.*, 1995; Kim *et al.*, 2003; Liu *et al.*, 2009; Lupold *et*

design that may elicit broadly neutralizing antibodies (Burton & Weiss, 2010).

Mechanism of

Interfere with gp120-CD4 binding

Interfere with binding of gp120 to CCR5 chemokine receptor and DC-SIGN. Also prevent loss of CD4+ T cells.

Interfere with HIV-1 fusion and induces shedding of gp120.

**7. Point mutations and anti-gp120 aptamers**

Fig. 8. Ribbon diagram illustrating epitopes of the gp120 core, highlighting some amino acid residues that interact with broadly neutralizing antibodies. The CD4bs (red) and the CD4i co-receptor-binding surface (green) as well as residues implicated in 2G12 binding (blue) are depicted.

In a clinically relevant study, paediatric HIV-1 subtype C clinical isolates with K665S/R/N point mutation were found to be resistant to a 2F5 broadly neutralizing monoclonal antibody (Gray *et al.*, 2006). This study confirmed earlier findings that the amino acid residue K665 is crucial for neutralization by 2F5 (Binley *et al.*, 2004). In the same study, the Lynn Morris' group found out that all the paediatric HIV-1 subtype C clinical isolates analysed had a conserved 4E10 epitope (W672, F673, W680) consistent with their phenotypic sensitivity to the 4E10 monoclonal antibody (Gray et al., 2006). 4E10 recognizes an epitope containing the sequence NWF(D/N)IT (Zwick et al., 2001). Mutagenesis studies have shown that the amino acid residues W672, F673 and W680 are indispensable for recognition by 4E10 (Zwick et al., 2005). Indeed, a recently published study describing four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization has shown that three subjects had W680 polymorphism, including a W680G point mutation (Nakamura et al., 2011). The study demonstrated that W680G point mutation was necessary to confer 4E10 resistant phenotype (Nakamura et al., 2011). In another subject a W680R point mutation caused variable resistant to 4E10 (Nakamura et al., 2011). A fourth subject possessed a F673L point mutation also associated with 4E10 resistance (Nakamura et al., 2011). Earlier, an independent study conducted in South Africa also showed that an HIV-1 subtype C virus isolated from a 7-year-old

Fig. 8. Ribbon diagram illustrating epitopes of the gp120 core, highlighting some amino acid residues that interact with broadly neutralizing antibodies. The CD4bs (red) and the CD4i co-receptor-binding surface (green) as well as residues implicated in 2G12 binding (blue) are

In a clinically relevant study, paediatric HIV-1 subtype C clinical isolates with K665S/R/N point mutation were found to be resistant to a 2F5 broadly neutralizing monoclonal antibody (Gray *et al.*, 2006). This study confirmed earlier findings that the amino acid residue K665 is crucial for neutralization by 2F5 (Binley *et al.*, 2004). In the same study, the Lynn Morris' group found out that all the paediatric HIV-1 subtype C clinical isolates analysed had a conserved 4E10 epitope (W672, F673, W680) consistent with their phenotypic sensitivity to the 4E10 monoclonal antibody (Gray et al., 2006). 4E10 recognizes an epitope containing the sequence NWF(D/N)IT (Zwick et al., 2001). Mutagenesis studies have shown that the amino acid residues W672, F673 and W680 are indispensable for recognition by 4E10 (Zwick et al., 2005). Indeed, a recently published study describing four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization has shown that three subjects had W680 polymorphism, including a W680G point mutation (Nakamura et al., 2011). The study demonstrated that W680G point mutation was necessary to confer 4E10 resistant phenotype (Nakamura et al., 2011). In another subject a W680R point mutation caused variable resistant to 4E10 (Nakamura et al., 2011). A fourth subject possessed a F673L point mutation also associated with 4E10 resistance (Nakamura et al., 2011). Earlier, an independent study conducted in South Africa also showed that an HIV-1 subtype C virus isolated from a 7-year-old

depicted.

perinatally infected child with F673L point mutation was resistant to 4E10 (Gray et al., 2008). These case studies are examples of the challenges presented by point mutations in controlling HIV-1 infection by antibodies. Notwithstanding, recent discovery of additional and more potent broadly neutralizing antibodies such as VRC01 (X. Wu *et al.*, 2010), PG9 and PG16 (Walker et al., 2009) is encouraging and boosts the impetus for HIV-1 vaccine design that may elicit broadly neutralizing antibodies (Burton & Weiss, 2010).

Table 8. Broadly neutralizing antibodies that bind gp120 and gp41 HIV-1 envelope proteins

#### **7. Point mutations and anti-gp120 aptamers**

Aptamers are synthetic oligonucleotide ligands that can be isolated *in vitro* against diverse targets including HIV-1 gp120 (Khati *et al.*, 2003). Aptamers assume a defined threedimensional structure and generally bind functional sites on their respective targets. They possess the molecular recognition properties of monoclonal antibodies in terms of their high affinity and specificity (Khati, 2010). Unlike antibodies, aptamers can fold properly and retain activity in the intracellular environment. However, the majority of aptamers with potential therapeutic utility selected to date target extracellular proteins (Chen *et al.*, 2003; Eyetech Study Group, 2002, 2003; Green *et al.*, 1995; Kim *et al.*, 2003; Liu *et al.*, 2009; Lupold *et*

Point Mutations Associated with HIV-1 Drug

or G. *Aids,* 19(16), 1799-1806.

18(12), 835-838.

357(6373), 85-89.

*J Virol,* 75(11), 4999-5008.

HIV-1 [letter]. *Nature,* 391(6664), 240.

neutralising antibodies.

**8. References**

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 113

bode well for the future development of aptamers such as B40 as an entry inhibitor drug, which will hopefully help circumvent HIV-1 point mutations and delay or diminish drug resistance. In order to maximally suppress or eradicate the virus; the aptamer or any other anti-HIV molecule in development should be used in combination with current antiretroviral drugs and the envisaged vaccines currently developed to elicit broadly

Abecasis, A. B., Deforche, K., Snoeck, J., Bacheler, L. T., McKenna, P., Carvalho, A. P.,

Antinori, A., Trotta, M. P., Lorenzini, P., Torti, C., Gianotti, N., Maggiolo, F., Ceccherini-

Aquaro, S., D'Arrigo, R., Svicher, V., Perri, G. D., Caputo, S. L., Visco-Comandini, U.,

receiving enfuvirtide treatment. *J Antimicrob Chemother,* 58(4), 714-722. Arion, D., Kaushik, N., McCormick, S., Borkow, G., & Parniak, M. A. (1998). Phenotypic

mutant viral reverse transcriptase. *Biochemistry,* 37(45), 15908-15917. Arnold, E., Jacobo-Molina, A., Nanni, R. G., Williams, R. L., Lu, X., Ding, J., Clark, A. D., Jr.,

Gomes, P., Camacho, R. J., & Vandamme, A. M. (2005). Protease mutation M89I/V is linked to therapy failure in patients infected with the HIV-1 non-B subtypes C, F

Silberstein, F., Nasto, P., Castagna, A., De Luca, A., Mussini, C., Andreoni, M., & Perno, C. F. (2007). Virological response to salvage therapy in HIV-infected persons carrying the reverse transcriptase K65R mutation. *Antivir Ther,* 12(8), 1175-1183. Antinori, A., Zaccarelli, M., Cingolani, A., Forbici, F., Rizzo, M. G., Trotta, M. P., Di

Giambenedetto, S., Narciso, P., Ammassari, A., Girardi, E., De Luca, A., & Perno, C. F. (2002). Cross-resistance among nonnucleoside reverse transcriptase inhibitors limits recycling efavirenz after nevirapine failure. *AIDS Res Hum Retroviruses,*

Santoro, M., Bertoli, A., Mazzotta, F., Bonora, S., Tozzi, V., Bellagamba, R., Zaccarelli, M., Narciso, P., Antinori, A., & Perno, C. F. (2006). Specific mutations in HIV-1 gp41 are associated with immunological success in HIV-1-infected patients

mechanism of HIV-1 resistance to 3'-azido-3'-deoxythymidine (AZT): increased polymerization processivity and enhanced sensitivity to pyrophosphate of the

Zhang, A., Ferris, A. L., Clark, P., & et al. (1992). Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations. *Nature,*

K., Larder, B., Buckery, R., Baker, D., Gallagher, K., Scarnati, H., Tritch, R., & Rizzo, C. (2001). Genotypic correlates of phenotypic resistance to efavirenz in virus isolates from patients failing nonnucleoside reverse transcriptase inhibitor therapy.

Mayers, D. L. (2006). Genotypic changes in human immunodeficiency virus type 1 protease associated with reduced susceptibility and virologic response to the

Q. J., Schuitemaker, H., Sodroski, J., & Weiss, R. A. (1998). A new classification for

Wilson, I. A., Elder, J. H., & de Parseval, A. (2006). Inhibition of HIV Env binding to

Bacheler, L., Jeffrey, S., Hanna, G., D'Aquila, R., Wallace, L., Logue, K., Cordova, B., Hertogs,

Baxter, J. D., Schapiro, J. M., Boucher, C. A., Kohlbrenner, V. M., Hall, D. B., Scherer, J. R., &

Berger, E. A., Doms, R. W., Fenyo, E. M., Korber, B. T., Littman, D. R., Moore, J. P., Sattentau,

Binley, J. M., Ngo-Abdalla, S., Moore, P., Bobardt, M., Chatterji, U., Gallay, P., Burton, D. R.,

protease inhibitor tipranavir. *J Virol,* 80(21), 10794-10801.

Fig. 9. A ribbon diagram of gp120 highlighting amino acid residues that interact with B40 aptamer. Residues in **bold** and single asterix are highly conserved among all HIV-1 isolates and significantly reduced binding of the aptamer by more than 10-fold when experimentally mutated to alanine, respectively (Joubert *et al.*, 2010). Those residues with double or tripple asterix are moderately and significantly varaiable, respectively, in all HIV isolates and while they make contact with the aptamer they did not significantly affect binding of the aptamer when experimentally mutated to alanine, respectively.

*al.*, 2002; Mi *et al.*, 2009; Nobile *et al.*, 1998; Ruckman *et al.*, 1998; Rusconi *et al.*, 2004; Rusconi *et al.*, 2002; Vinores, 2003; White *et al.*, 2003).

Extracellular therapeutic targets such as HIV-1 gp120 have the advantage of ready access to aptamer intervention without the need for enabling access to cells. The aptamers against HIV-1 gp120 bound the protein with high affinity, high specificity and neutralized a broad range of R5 HIV-1 clinical isolates (Khati *et al.*, 2003). In a recent review, these RNA aptamers were reported to have the most potent in vitro antiviral efficacy of all HIV-1 entry inhibitors described to date (Held *et al.*, 2006), including antibodies. These aptamers prevented entry and suppressed viral replication in cultured human peripheral blood mononuclear cells (PBMC) by up to 10,000-fold (Khati *et al.*, 2003). While the emergence of HIV-1 escape mutants and drug resistance variants appears to be inevitable, our goup and collaborators have recently shown that one extensively studied aptamer called B40 penetrate the highly variable exterior surfaces of gp120 and bind the conserved core at the heart of the CCR5-binding site (Cohen *et al.*, 2008; Dey *et al.*, 2005; Joubert *et al.*, 2010), which the virus may be unable to mutate without compromising its fitness. Four amino acids (Q114, K117, K207, and I439) of gp120 (Figure 9) that weakened binding of gp120 to B40 aptamer by at least 10-fold when experimentally mutated to alanine (Joubert et al., 2010) are naturally conserved among all HIV-1 isolates (Kwong et al., 1998a). This suggests that B40 makes direct contact with at least four conserved core residues on gp120 within the CCR5-binding site, which the virus cannot afford to mutate without losing selective advantage. These data bode well for the future development of aptamers such as B40 as an entry inhibitor drug, which will hopefully help circumvent HIV-1 point mutations and delay or diminish drug resistance. In order to maximally suppress or eradicate the virus; the aptamer or any other anti-HIV molecule in development should be used in combination with current antiretroviral drugs and the envisaged vaccines currently developed to elicit broadly neutralising antibodies.

#### **8. References**

112 Point Mutation

Fig. 9. A ribbon diagram of gp120 highlighting amino acid residues that interact with B40 aptamer. Residues in **bold** and single asterix are highly conserved among all HIV-1 isolates and significantly reduced binding of the aptamer by more than 10-fold when experimentally mutated to alanine, respectively (Joubert *et al.*, 2010). Those residues with double or tripple asterix are moderately and significantly varaiable, respectively, in all HIV isolates and while they make contact with the aptamer they did not significantly affect binding of the aptamer

*al.*, 2002; Mi *et al.*, 2009; Nobile *et al.*, 1998; Ruckman *et al.*, 1998; Rusconi *et al.*, 2004; Rusconi

Extracellular therapeutic targets such as HIV-1 gp120 have the advantage of ready access to aptamer intervention without the need for enabling access to cells. The aptamers against HIV-1 gp120 bound the protein with high affinity, high specificity and neutralized a broad range of R5 HIV-1 clinical isolates (Khati *et al.*, 2003). In a recent review, these RNA aptamers were reported to have the most potent in vitro antiviral efficacy of all HIV-1 entry inhibitors described to date (Held *et al.*, 2006), including antibodies. These aptamers prevented entry and suppressed viral replication in cultured human peripheral blood mononuclear cells (PBMC) by up to 10,000-fold (Khati *et al.*, 2003). While the emergence of HIV-1 escape mutants and drug resistance variants appears to be inevitable, our goup and collaborators have recently shown that one extensively studied aptamer called B40 penetrate the highly variable exterior surfaces of gp120 and bind the conserved core at the heart of the CCR5-binding site (Cohen *et al.*, 2008; Dey *et al.*, 2005; Joubert *et al.*, 2010), which the virus may be unable to mutate without compromising its fitness. Four amino acids (Q114, K117, K207, and I439) of gp120 (Figure 9) that weakened binding of gp120 to B40 aptamer by at least 10-fold when experimentally mutated to alanine (Joubert et al., 2010) are naturally conserved among all HIV-1 isolates (Kwong et al., 1998a). This suggests that B40 makes direct contact with at least four conserved core residues on gp120 within the CCR5-binding site, which the virus cannot afford to mutate without losing selective advantage. These data

when experimentally mutated to alanine, respectively.

*et al.*, 2002; Vinores, 2003; White *et al.*, 2003).

Point Mutations Associated with HIV-1 Drug

*Virol,* 66(12), 7517-7521.

355-365.

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 115

Clark, S. A., Shulman, N. S., Bosch, R. J., & Mellors, J. W. (2006). Reverse transcriptase

Cohen, C., Forzan, M., Sproat, B., Pantophlet, R., McGowan, I., Burton, D., & James, W.

Collman, R., Balliet, J. W., Gregory, S. A., Friedman, H., Kolson, D. L., Nathanson, N., &

Colonno, R., Rose, R., McLaren, C., Thiry, A., Parkin, N., & Friborg, J. (2004). Identification of

Cooper, D. A., Steigbigel, R. T., Gatell, J. M., Rockstroh, J. K., Katlama, C., Yeni, P., Lazzarin,

Corti, D., Langedijk, J. P., Hinz, A., Seaman, M. S., Vanzetta, F., Fernandez-Rodriguez, B. M.,

Cotton, M. F., Ikle, D. N., Rapaport, E. L., Marschner, S., Tseng, P. O., Kurrle, R., & Finkel, T.

Cozzi-Lepri, A., Ruiz, L., Loveday, C., Phillips, A. N., Clotet, B., Reiss, P., Ledergerber, B.,

impact on the virological response to therapy. *Antivir Ther,* 10(7), 791-802. Dandache, S., Sevigny, G., Yelle, J., Stranix, B. R., Parkin, N., Schapiro, J. M., Wainberg, M.

Das, K., Bauman, J. D., Clark, A. D., Jr., Frenkel, Y. V., Lewi, P. J., Shatkin, A. J., Hughes, S.

Das, K., Clark, A. D., Jr., Lewi, P. J., Heeres, J., De Jonge, M. R., Koymans, L. M., Vinkers, H.

resistance mutations. *Proc Natl Acad Sci U S A,* 105(5), 1466-1471.

reverse transcriptase inhibitors. *Aids,* 20(7), 981-984.

gp120 in the CCR5 binding site. *Virology,* 381(1), 46-54.

infected individuals. *J Exp Med,* 185(4), 621-628.

infected individuals. *PLoS One,* 5(1), e8805.

*Antimicrob Agents Chemother,* 51(11), 4036-4043.

*Pediatr Res,* 42(5), 656-664.

mutations 118I, 208Y, and 215Y cause HIV-1 hypersusceptibility to non-nucleoside

(2008). An aptamer that neutralizes R5 strains of HIV-1 binds to core residues of

Srinivasan, A. (1992). An infectious molecular clone of an unusual macrophagetropic and highly cytopathic strain of human immunodeficiency virus type 1. *J*

I50L as the signature atazanavir (ATV)-resistance mutation in treatment-naive HIV-1 infected patients receiving ATV-containing regimens. *J Infect Dis,* 189(10), 1802-1810. Connor, R. I., Sheridan, K. E., Ceradini, D., Choe, S., & Landau, N. R. (1997). Change in

coreceptor use coreceptor use correlates with disease progression in HIV-1--

A., Clotet, B., Kumar, P. N., Eron, J. E., Schechter, M., Markowitz, M., Loutfy, M. R., Lennox, J. L., Zhao, J., Chen, J., Ryan, D. M., Rhodes, R. R., Killar, J. A., Gilde, L. R., Strohmaier, K. M., Meibohm, A. R., Miller, M. D., Hazuda, D. J., Nessly, M. L., DiNubile, M. J., Isaacs, R. D., Teppler, H., & Nguyen, B. Y. (2008). Subgroup and resistance analyses of raltegravir for resistant HIV-1 infection. *N Engl J Med,* 359(4),

Silacci, C., Pinna, D., Jarrossay, D., Balla-Jhagjhoorsingh, S., Willems, B., Zekveld, M. J., Dreja, H., O'Sullivan, E., Pade, C., Orkin, C., Jeffs, S. A., Montefiori, D. C., Davis, D., Weissenhorn, W., McKnight, A., Heeney, J. L., Sallusto, F., Sattentau, Q. J., Weiss, R. A., & Lanzavecchia, A. (2010). Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-

H. (1997). Apoptosis of CD4+ and CD8+ T cells isolated immediately ex vivo correlates with disease severity in human immunodeficiency virus type 1 infection.

Holkmann, C., Staszewski, S., & Lundgren, J. D. (2005). Thymidine analogue mutation profiles: factors associated with acquiring specific profiles and their

A., & Wu, J. J. (2007). In vitro antiviral activity and cross-resistance profile of PL-100, a novel protease inhibitor of human immunodeficiency virus type 1.

H., & Arnold, E. (2008). High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: strategic flexibility explains potency against

M., Daeyaert, F., Ludovici, D. W., Kukla, M. J., De Corte, B., Kavash, R. W., Ho, C. Y., Ye, H., Lichtenstein, M. A., Andries, K., Pauwels, R., De Bethune, M. P., Boyer,

cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120. *Retrovirology,* 3, 39.

Binley, J. M., Wrin, T., Korber, B., Zwick, M. B., Wang, M., Chappey, C., Stiegler, G., Kunert,

Boyer, P. L., Sarafianos, S. G., Arnold, E., & Hughes, S. H. (2001). Selective excision of

Brenner, B. G., Oliveira, M., Doualla-Bell, F., Moisi, D. D., Ntemgwa, M., Frankel, F., Essex,

Brun-Vezinet, F., Descamps, D., Ruffault, A., Masquelier, B., Calvez, V., Peytavin, G., Telles, F.,

Cabrera, C., Marfil, S., Garcia, E., Martinez-Picado, J., Bonjoch, A., Bofill, M., Moreno, S.,

long-term enfuvirtide-containing salvage regimen. *Aids,* 20(16), 2075-2080. Calarese, D. A., Scanlan, C. N., Zwick, M. B., Deechongkit, S., Mimura, Y., Kunert, R., Zhu,

Calazans, A., Brindeiro, R., Brindeiro, P., Verli, H., Arruda, M. B., Gonzalez, L. M.,

Ceccherini-Silberstein, F., Erba, F., Gago, F., Bertoli, A., Forbici, F., Bellocchi, M. C., Gori, C.,

Charpentier, C., Jenabian, M. A., Piketty, C., Karmochkine, M., Tisserand, P., Laureillard, D.,

Chen, C. H., Chernis, G. A., Hoang, V. Q., & Landgraf, R. (2003). Inhibition of heregulin

caused by the L89M polymorphism. *J Infect Dis,* 191(11), 1961-1970. Cane, P. A., de Ruiter, A., Rice, P., Wiselka, M., Fox, R., & Pillay, D. (2001). Resistance-

presence and absence of drug pressure. *Aids,* 18(12), F11-19.

under salvage therapy. *Scand J Infect Dis,* 43(5), 373-379.

resistance to tenofovir in cell culture. *Aids,* 20(9), F9-13.

*Retrovirology,* 3, 39.

*Virol,* 75(10), 4832-4842.

329(5993), 770-773.

2065-2071.

*Microbiol,* 39(7), 2652-2654.

*Cell,* 85(7), 1135-1148.

cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120.

R., Zolla-Pazner, S., Katinger, H., Petropoulos, C. J., & Burton, D. R. (2004). Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies. *J Virol,* 78(23), 13232-13252.

AZTMP by drug-resistant human immunodeficiency virus reverse transcriptase. *J*

M., & Wainberg, M. A. (2006). HIV-1 subtype C viruses rapidly develop K65R

Morand-Joubert, L., Meynard, J. L., Vray, M., & Costagliola, D. (2003). Clinically relevant interpretation of genotype for resistance to abacavir. *Aids,* 17(12), 1795-1802. Burton, D. R., & Weiss, R. A. (2010). AIDS/HIV. A boost for HIV vaccine design. *Science,*

Ribera, E., Domingo, P., Clotet, B., & Ruiz, L. (2006). Genetic evolution of gp41 reveals a highly exclusive relationship between codons 36, 38 and 43 in gp41 under

P., Wormald, M. R., Stanfield, R. L., Roux, K. H., Kelly, J. W., Rudd, P. M., Dwek, R. A., Katinger, H., Burton, D. R., & Wilson, I. A. (2003). Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. *Science,* 300(5628),

Guimaraes, J. A., Diaz, R. S., Antunes, O. A., & Tanuri, A. (2005). Low accumulation of L90M in protease from subtype F HIV-1 with resistance to protease inhibitors is

associated mutations in the human immunodeficiency virus type 1 subtype c protease gene from treated and untreated patients in the United Kingdom. *J Clin*

D'Arrigo, R., Marcon, L., Balotta, C., Antinori, A., Monforte, A. D., & Perno, C. F. (2004). Identification of the minimal conserved structure of HIV-1 protease in the

Belec, L., Si-Mohamed, A., & Weiss, L. (2011). Dynamics of enfuvirtide resistance mutations in enfuvirtide-experienced patients remaining in virological failure

signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3. *Proc Natl Acad Sci U S A,* 100(16), 9226-9231. Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., Wu, L., Mackay, C. R.,

LaRosa, G., Newman, W., Gerard, N., Gerard, C., & Sodroski, J. (1996). The betachemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.

Point Mutations Associated with HIV-1 Drug

*Antivir Ther,* 11(6), 701-705.

type 1. *J Virol,* 71(9), 6305-6314.

cofactors. *Cell,* 85(7), 1149-1158.

*Nature,* 381(6584), 667-673.

trial. *Lancet,* 368(9534), 476-482.

*Acquir Immune Defic Syndr,* 45(2), 249-251.

degeneration. *Retina,* 22(2), 143-152.

interaction. *J Virol,* 79(21), 13806-13810.

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 117

Descamps, D., Ait-Khaled, M., Craig, C., Delarue, S., Damond, F., Collin, G., & Brun-

Dey, A. K., Khati, M., Tang, M., Wyatt, R., Lea, S. M., & James, W. (2005). An aptamer that

di Marzo Veronese, F., Copeland, T. D., DeVico, A. L., Rahman, R., Oroszlan, S., Gallo, R. C.,

the reverse transcriptase of HTLV-III/LAV. *Science,* 231(4743), 1289-1291. Doranz, B. J., Lu, Z. H., Rucker, J., Zhang, T. Y., Sharron, M., Cen, Y. H., Wang, Z. X., Guo,

Doranz, B. J., Rucker, J., Yi, Y., Smyth, R. J., Samson, M., Peiper, S. C., Parmentier, M.,

Doualla-Bell, F., Avalos, A., Brenner, B., Gaolathe, T., Mine, M., Gaseitsiwe, S., Oliveira, M.,

Dragic, T., Litwin, V., Allaway, G. P., Martin, S. R., Huang, Y., Nagashima, K. A., Cayanan,

Dragic, T., Trkola, A., Thompson, D. A., Cormier, E. G., Kajumo, F. A., Maxwell, E., Lin, S.

Eron, J., Jr., Yeni, P., Gathe, J., Jr., Estrada, V., DeJesus, E., Staszewski, S., Lackey, P.,

Eron, J. J., Jr., Bosch, R. J., Bettendorf, D., Petch, L., Fiscus, S., & Frank, I. (2007). The effect of

Euler, Z., Bunnik, E. M., Burger, J. A., Boeser-Nunnink, B. D., Grijsen, M. L., Prins, J. M., &

Eyetech Study Group (2002). Preclinical and phase 1A clinical evaluation of an anti-VEGF

from early and late in the epidemic. *J Virol,* 85(14), 7236-7245.

regimens. *Antimicrob Agents Chemother,* 50(12), 4182-4185.

CCR5. *Proc Natl Acad Sci U S A,* 97(10), 5639-5644.

HIV-1 [see comments]. *Nature,* 381(6584), 661-666.

Landau, N. R. (1996). Identification of a major co-receptor for primary isolates of

Vezinet, F. (2006). Rare selection of the K65R mutation in antiretroviral-naive patients failing a first-line abacavir/ lamivudine-containing HAART regimen.

neutralizes R5 strains of human immunodeficiency virus type 1 blocks gp120-CCR5

& Sarngadharan, M. G. (1986). Characterization of highly immunogenic p66/p51 as

H. H., Du, J. G., Accavitti, M. A., Doms, R. W., & Peiper, S. C. (1997). Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus

Collman, R. G., & Doms, R. W. (1996). A dual-tropic primary HIV-1 isolate that uses fusin and the beta- chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion

Moisi, D., Ndwapi, N., Moffat, H., Essex, M., & Wainberg, M. A. (2006). High prevalence of the K65R mutation in human immunodeficiency virus type 1 subtype C isolates from infected patients in Botswana treated with didanosine-based

C., Maddon, P. J., Koup, R. A., Moore, J. P., & Paxton, W. A. (1996). HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC- CKR-5 [see comments].

W., Ying, W., Smith, S. O., Sakmar, T. P., & Moore, J. P. (2000). A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of

Katlama, C., Young, B., Yau, L., Sutherland-Phillips, D., Wannamaker, P., Vavro, C., Patel, L., Yeo, J., & Shaefer, M. (2006). The KLEAN study of fosamprenavirritonavir versus lopinavir-ritonavir, each in combination with abacavir-lamivudine, for initial treatment of HIV infection over 48 weeks: a randomised non-inferiority

lamivudine therapy and M184V on the antiretroviral activity of didanosine. *J*

Schuitemaker, H. (2011). Activity of broadly neutralizing antibodies, including PG9, PG16, and VRC01, against recently transmitted subtype B HIV-1 variants

pegylated aptamer (EYE001) for the treatment of exudative age-related macular

P. L., Clark, P., Hughes, S. H., Janssen, P. A., & Arnold, E. (2004). Roles of conformational and positional adaptability in structure-based design of TMC125- R165335 (etravirine) and related non-nucleoside reverse transcriptase inhibitors that are highly potent and effective against wild-type and drug-resistant HIV-1 variants. *J Med Chem,* 47(10), 2550-2560.

Das, K., Ding, J., Hsiou, Y., Clark, A. D., Jr., Moereels, H., Koymans, L., Andries, K.,

De Luca, A., Giambenedetto, S. D., Trotta, M. P., Colafigli, M., Prosperi, M., Ruiz, L., Baxter, J.,

Deforche, K., Camacho, R., Grossman, Z., Silander, T., Soares, M. A., Moreau, Y., Shafer, R.

pathways against HIV-1 protease inhibitors. *Infect Genet Evol,* 7(3), 382-390.  *J Infect Dis,* 193(9), 1219-1222.

variants. *J Med Chem,* 47(10), 2550-2560.

*J Infect Dis,* 195(3), 387-391.

*Bioinformatics,* 22(24), 2975-2979.

regimen. *J Med Virol,* 65(3), 445-448.

*HIV Clin Trials,* 9(1), 11-25.

*Antimicrob Agents Chemother,* 54(1), 491-501.

P. L., Clark, P., Hughes, S. H., Janssen, P. A., & Arnold, E. (2004). Roles of conformational and positional adaptability in structure-based design of TMC125- R165335 (etravirine) and related non-nucleoside reverse transcriptase inhibitors that are highly potent and effective against wild-type and drug-resistant HIV-1

Pauwels, R., Janssen, P. A., Boyer, P. L., Clark, P., Smith, R. H., Jr., Kroeger Smith, M. B., Michejda, C. J., Hughes, S. H., & Arnold, E. (1996). Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant. *J Mol Biol,* 264(5), 1085-1100. De Luca, A., Di Giambenedetto, S., Romano, L., Gonnelli, A., Corsi, P., Baldari, M., Di Pietro,

M., Menzo, S., Francisci, D., Almi, P., & Zazzi, M. (2006). Frequency and treatmentrelated predictors of thymidine-analogue mutation patterns in HIV-1 isolates after

Clevenbergh, P., Cauda, R., Perno, C. F., & Antinori, A. (2007). Improved interpretation of genotypic changes in the HIV-1 reverse transcriptase coding region that determine the virological response to didanosine. *J Infect Dis,* 196(11), 1645-1653. Deeks, S. G., Lu, J., Hoh, R., Neilands, T. B., Beatty, G., Huang, W., Liegler, T., Hunt, P.,

Martin, J. N., & Kuritzkes, D. R. (2007). Interruption of enfuvirtide in HIV-1 infected adults with incomplete viral suppression on an enfuvirtide-based regimen.

W., Van Laethem, K., Carvalho, A. P., Wynhoven, B., Cane, P., Snoeck, J., Clarke, J., Sirivichayakul, S., Ariyoshi, K., Holguin, A., Rudich, H., Rodrigues, R., Bouzas, M. B., Cahn, P., Brigido, L. F., Soriano, V., Sugiura, W., Phanuphak, P., Morris, L., Weber, J., Pillay, D., Tanuri, A., Harrigan, P. R., Shapiro, J. M., Katzenstein, D. A., Kantor, R., & Vandamme, A. M. (2007). Bayesian network analysis of resistance

Kantor, R., Moreau, Y., & Vandamme, A. M. (2006). Analysis of HIV-1 pol sequences using Bayesian Networks: implications for drug resistance.

Katlama, C., & Calvez, V. (2001). Resistance profile and cross-resistance of HIV-1 among patients failing a non-nucleoside reverse transcriptase inhibitor-containing

Deprez, E., Marcelin, A. G., Tchertanov, L., & Mouscadet, J. F. (2011). Impact of Y143 HIV-1 integrase mutations on resistance to raltegravir in vitro and in vivo.

Spreen, W., Nguyen, B. Y., Koval, C. E., Eron, J. J., Hammer, S., & Squires, K. (2008). Association of efavirenz hypersusceptibility with virologic response in ACTG 368, a randomized trial of abacavir (ABC) in combination with efavirenz (EFV) and indinavir (IDV) in HIV-infected subjects with prior nucleoside analog experience.

S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R., &

Landau, N. R. (1996). Identification of a major co-receptor for primary isolates of HIV-1 [see comments]. *Nature,* 381(6584), 661-666.

Point Mutations Associated with HIV-1 Drug

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 119

Green, L. S., Jellinek, D., Bell, C., Beebe, L. A., Feistner, B. D., Gill, S. C., Jucker, F. M., &

Harrigan, P. R., Stone, C., Griffin, P., Najera, I., Bloor, S., Kemp, S., Tisdale, M., & Larder, B.

Hawkins, C. A., Chaplin, B., Idoko, J., Ekong, E., Adewole, I., Gashau, W., Murphy, R. L., &

Held, D. M., Kissel, J. D., Patterson, J. T., Nickens, D. G., & Burke, D. H. (2006). HIV-1

Herbein, G., Mahlknecht, U., Batliwalla, F., Gregersen, P., Pappas, T., Butler, J., O'Brien, W.

Ho, D. D., McKeating, J. A., Li, X. L., Moudgil, T., Daar, E. S., Sun, N. C., & Robinson, J. E.

Hoffman, N. G., Schiffer, C. A., & Swanstrom, R. (2003). Covariation of amino acid positions

Hombrouck, A., Voet, A., Van Remoortel, B., Desadeleer, C., De Maeyer, M., Debyser, Z., &

Hsiou, Y., Ding, J., Das, K., Clark, A. D., Jr., Boyer, P. L., Lewi, P., Janssen, P. A., Kleim, J. P.,

RT: a novel mechanism of drug resistance. *J Mol Biol,* 309(2), 437-445. Huang, K. H., Goedhals, D., Carlson, J. M., Brockman, M. A., Mishra, S., Brumme, Z. L.,

reverting and compensatory viral mutations. *PLoS One,* 6(4), e19018.

factor/vascular endothelial growth factor. *Chem Biol,* 2(10), 683-695. Grossman, Z., Paxinos, E. E., Averbuch, D., Maayan, S., Parkin, N. T., Engelhard, D., Lorber,

therapy. CNA2001 Investigative Group. *J Infect Dis,* 181(3), 912-920. Hartley, O., Klasse, P. J., Sattentau, Q. J., & Moore, J. P. (2005). V3: HIV's switch-hitter. *AIDS*

inactivation by nucleic acid aptamers. *Front Biosci,* 11, 89-112.

neutralizing antibody response. *J Virol,* 82(5), 2367-2375.

nelfinavir. *Antimicrob Agents Chemother,* 48(6), 2159-2165.

*Res Hum Retroviruses,* 21(2), 171-189.

*Defic Syndr,* 52(2), 228-234.

antibody. *J Virol,* 65(1), 489-493.

in HIV-1 protease. *Virology,* 314(2), 536-548.

function. *J Biol Chem,* 274(23), 16228-16234.

395(6698), 189-194.

1 subtype C-infected individual with an anti-membrane-proximal external region-

Janjic, N. (1995). Nuclease-resistant nucleic acid ligands to vascular permeability

M., Istomin, V., Shaked, Y., Mendelson, E., Ram, D., Petropoulos, C. J., & Schapiro, J. M. (2004). Mutation D30N is not preferentially selected by human immunodeficiency virus type 1 subtype C in the development of resistance to

(2000). Resistance profile of the human immunodeficiency virus type 1 reverse transcriptase inhibitor abacavir (1592U89) after monotherapy and combination

Kanki, P. (2009). Clinical and genotypic findings in HIV-infected patients with the K65R mutation failing first-line antiretroviral therapy in Nigeria. *J Acquir Immune*

A., & Verdin, E. (1998). Apoptosis of CD8+ T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4. *Nature,*

(1991). Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal

Witvrouw, M. (2008). Mutations in human immunodeficiency virus type 1 integrase confer resistance to the naphthyridine L-870,810 and cross-resistance to the clinical trial drug GS-9137. *Antimicrob Agents Chemother,* 52(6), 2069-2078. Howard, O. M., Shirakawa, A. K., Turpin, J. A., Maynard, A., Tobin, G. J., Carrington, M.,

Oppenheim, J. J., & Dean, M. (1999). Naturally occurring CCR5 extracellular and transmembrane domain variants affect HIV-1 Co-receptor and ligand binding

Rosner, M., Hughes, S. H., & Arnold, E. (2001). The Lys103Asn mutation of HIV-1

Hickling, S., Tang, C. S., Miura, T., Seebregts, C., Heckerman, D., Ndung'u, T., Walker, B., Klenerman, P., Steyn, D., Goulder, P., Phillips, R., van Vuuren, C., & Frater, J. (2011). Progression to AIDS in South Africa is associated with both

Eyetech Study Group (2003). Anti-vascular endothelial growth factor therapy for subfoveal

Fischl, M. A., Richman, D. D., Grieco, M. H., Gottlieb, M. S., Volberding, P. A., Laskin, O. L.,

Frater, A. J., Chaput, C. C., Beddows, S., Weber, J. N., & McClure, M. O. (2001). Simple

Frey, G., Peng, H., Rits-Volloch, S., Morelli, M., Cheng, Y., & Chen, B. (2008). A fusion-

Friend, J., Parkin, N., Liegler, T., Martin, J. N., & Deeks, S. G. (2004). Isolated lopinavir

Gallant, J. E., DeJesus, E., Arribas, J. R., Pozniak, A. L., Gazzard, B., Campo, R. E., Lu, B.,

Gallant, J. E., Staszewski, S., Pozniak, A. L., DeJesus, E., Suleiman, J. M., Miller, M. D.,

Gao, H. Q., Boyer, P. L., Sarafianos, S. G., Arnold, E., & Hughes, S. H. (2000). The role of

Garg, H., Joshi, A., Freed, E. O., & Blumenthal, R. (2007). Site-specific mutations in HIV-1

Gilead Sciences (2008). Phase III trial begins for elvitegravir. *AIDS Patient Care STDS,* 22(9),

Gonzales, M. J., Wu, T. D., Taylor, J., Belitskaya, I., Kantor, R., Israelski, D., Chou, S., Zolopa,

Gonzalez, L. M., Brindeiro, R. M., Aguiar, R. S., Pereira, H. S., Abreu, C. M., Soares, M. A., &

subtype B and C proteases. *Antimicrob Agents Chemother,* 48(9), 3552-3555. Gougeon, M. L., & Montagnier, L. (1993). Apoptosis in AIDS. *Science,* 260(5112), 1269-1270. Gray, E. S., Meyers, T., Gray, G., Montefiori, D. C., & Morris, L. (2006). Insensitivity of

Gray, E. S., Moore, P. L., Bibollet-Ruche, F., Li, H., Decker, J. M., Meyers, T., Shaw, G. M., &

FDA (2007). FDA approves raltegravir tablets. *AIDS Patient Care STDS,* 21(11), 889.

II study results. *Ophthalmology,* 110(5), 979-986.

*Natl Acad Sci U S A,* 105(10), 3739-3744.

3-year randomized trial. *Jama,* 292(2), 191-201.

fusion/hemifusion. *J Biol Chem,* 282(23), 16899-16906.

raised against subtype B. *PLoS Med,* 3(7), e255.

transcriptase. *J Mol Biol,* 300(2), 403-418.

191.

93(1-2), 145-156.

18(14), 1965-1966.

762-763.

*Aids,* 17(6), 791-799.

*Engl J Med,* 354(3), 251-260.

choroidal neovascularization secondary to age-related macular degeneration: phase

Leedom, J. M., Groopman, J. E., Mildvan, D., Schooley, R. T., & et al. (1987). The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDSrelated complex. A double-blind, placebo-controlled trial. *N Engl J Med,* 317(4), 185-

detection of point mutations associated with HIV-1 drug resistance. *J Virol Methods,*

intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies. *Proc*

resistance after virological rebound of a ritonavir/lopinavir-based regimen. *Aids,*

McColl, D., Chuck, S., Enejosa, J., Toole, J. J., & Cheng, A. K. (2006). Tenofovir DF, emtricitabine, and efavirenz vs. zidovudine, lamivudine, and efavirenz for HIV. *N*

Coakley, D. F., Lu, B., Toole, J. J., & Cheng, A. K. (2004). Efficacy and safety of tenofovir DF vs stavudine in combination therapy in antiretroviral-naive patients: a

steric hindrance in 3TC resistance of human immunodeficiency virus type-1 reverse

gp41 reveal a correlation between HIV-1-mediated bystander apoptosis and

A. R., Fessel, W. J., & Shafer, R. W. (2003). Extended spectrum of HIV-1 reverse transcriptase mutations in patients receiving multiple nucleoside analog inhibitors.

Tanuri, A. (2004). Impact of nelfinavir resistance mutations on in vitro phenotype, fitness, and replication capacity of human immunodeficiency virus type 1 with

paediatric HIV-1 subtype C viruses to broadly neutralising monoclonal antibodies

Morris, L. (2008). 4E10-resistant variants in a human immunodeficiency virus type

1 subtype C-infected individual with an anti-membrane-proximal external regionneutralizing antibody response. *J Virol,* 82(5), 2367-2375.

Point Mutations Associated with HIV-1 Drug

12692-12698.

*Ther,* 9(1), 37-45.

*Ther,* 12(4), 563-570.

regimens. *HIV Med,* 5(6), 394-399.

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 121

transcriptase genotype: results of a global collaboration. *PLoS Med,* 2(4), e112.

Khati, M., Schuman, M., Ibrahim, J., Sattentau, Q., Gordon, S., & James, W. (2003).

Kilby, J. M., Hopkins, S., Venetta, T. M., DiMassimo, B., Cloud, G. A., Lee, J. Y., Alldredge, L.,

Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., & Steitz, T. A. (1992a). Crystal

Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., & Steitz, T. A. (1992b). Crystal

Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., & Hendrickson, W. A.

Lanier, E. R., Givens, N., Stone, C., Griffin, P., Gibb, D., Walker, S., Tisdale, M., Irlbeck, D.,

Lataillade, M., Chiarella, J., & Kozal, M. J. (2007). Natural polymorphism of the HIV-1

Lazzarin, A., Campbell, T., Clotet, B., Johnson, M., Katlama, C., Moll, A., Towner, W.,

randomised, double-blind, placebo-controlled trial. *Lancet,* 370(9581), 39-48. Li, C. J., Friedman, D. J., Wang, C., Metelev, V., & Pardee, A. B. (1995). Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein. *Science,* 268(5209), 429-431. Li, Y., O'Dell, S., Walker, L. M., Wu, X., Guenaga, J., Feng, Y., Schmidt, S. D., McKee, K.,

neutralizing HIV-1 monoclonal antibody VRC01. *J Virol,* 85(17), 8954-8967.

receptor and a neutralizing human antibody. *Nature,* 393(6686), 648-659. Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., & Hendrickson, W. A. (1998b).

Khati, M. (2010). The future of aptamers in medicine. *J Clin Pathol,* 63(6), 480-487.

*Biochem Biophys Res Commun,* 300(2), 516-523.

inhibitor. *Science,* 256(5065), 1783-1790.

inhibitor. *Science,* 256(5065), 1783-1790.

Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse

Neutralization of infectivity of diverse R5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2'F-RNA aptamers. *J Virol,* 77(23),

Hunter, E., Lambert, D., Bolognesi, D., Matthews, T., Johnson, M. R., Nowak, M. A., Shaw, G. M., & Saag, M. S. (1998). Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. *Nat Med,* 4(11), 1302-1307. Kim, Y. M., Choi, K. H., Jang, Y. J., Yu, J., & Jeong, S. (2003). Specific modulation of the anti-

DNA autoantibody-nucleic acids interaction by the high affinity RNA aptamer.

structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an

structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an

(1998a). Structure of an HIV gp120 envelope glycoprotein in complex with the CD4

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody [see comments]. *Nature,* 393(6686), 648-659. Lanier, E. R., Ait-Khaled, M., Scott, J., Stone, C., Melby, T., Sturge, G., St Clair, M., Steel, H.,

Hetherington, S., Pearce, G., Spreen, W., & Lafon, S. (2004a). Antiviral efficacy of abacavir in antiretroviral therapy-experienced adults harbouring HIV-1 with specific patterns of resistance to nucleoside reverse transcriptase inhibitors. *Antivir*

Underwood, M., St Clair, M., & Ait-Khaled, M. (2004b). Effect of concurrent zidovudine use on the resistance pathway selected by abacavir-containing

integrase gene and mutations associated with integrase inhibitor resistance. *Antivir*

Trottier, B., Peeters, M., Vingerhoets, J., de Smedt, G., Baeten, B., Beets, G., Sinha, R., & Woodfall, B. (2007). Efficacy and safety of TMC125 (etravirine) in treatmentexperienced HIV-1-infected patients in DUET-2: 24-week results from a

Louder, M. K., Ledgerwood, J. E., Graham, B. S., Haynes, B. F., Burton, D. R., Wyatt, R. T., & Mascola, J. R. (2011). Mechanism of neutralization by the broadly

Huang, W., Eshleman, S. H., Toma, J., Fransen, S., Stawiski, E., Paxinos, E. E., Whitcomb, J.

heterogeneous composition of viral populations. *J Virol,* 81(15), 7885-7893. Huang, W., Toma, J., Fransen, S., Stawiski, E., Reeves, J. D., Whitcomb, J. M., Parkin, N., &

Ishikawa, H., Sasaki, M., Noda, S., & Koga, Y. (1998). Apoptosis induction by the binding of

Janssen, P. A., Lewi, P. J., Arnold, E., Daeyaert, F., de Jonge, M., Heeres, J., Koymans, L.,

Johnson, A. A., Santos, W., Pais, G. C., Marchand, C., Amin, R., Burke, T. R., Jr., Verdine, G.,

Johnson, V. A., Brun-Vezinet, F., Clotet, B., Gunthard, H. F., Kuritzkes, D. R., Pillay, D.,

Johnston, E., Winters, M. A., Rhee, S. Y., Merigan, T. C., Schiffer, C. A., & Shafer, R. W.

Joshi, A., Nyakeriga, A., Ravi, R., & Garg, H. (2011). HIV ENV glycoprotein mediated

Joubert, M. K., Kinsley, N., Capovilla, A., Sewell, B. T., Jaffer, M. A., & Khati, M. (2010). A

Kalia, V., Sarkar, S., Gupta, P., & Montelaro, R. C. (2005). Antibody neutralization escape

Kantor, R., Katzenstein, D. A., Efron, B., Carvalho, A. P., Wynhoven, B., Cane, P., Clarke, J.,

mechanism of HIV-1 neutralization. *Biochemistry,* 49(28), 5880-5890. Kagan, R. M., Shenderovich, M. D., Heseltine, P. N., & Ramnarayan, K. (2005). Structural

immunodeficiency virus type 1 gp41. *J Virol,* 79(4), 2097-2107.

virus type 1 envelope protein. *J Virol,* 82(11), 5584-5593.

in HIV-1: December 2010. *Top HIV Med,* 18(5), 156-163.

resistance. *Antimicrob Agents Chemother,* 48(12), 4864-4868.

calmodulin. *J Virol,* 72(8), 6574-6580.

analysis. *AIDS Rev,* 5(2), 104-112.

env fusogenic activity. *J Biol Chem*.

lopinavir. *Protein Sci,* 14(7), 1870-1878.

*Chem,* 281(1), 461-467.

M., Young, A. M., Donnell, D., Mmiro, F., Musoke, P., Guay, L. A., Jackson, J. B., Parkin, N. T., & Petropoulos, C. J. (2007). Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and

Petropoulos, C. J. (2008). Coreceptor tropism can be influenced by amino acid substitutions in the gp41 transmembrane subunit of human immunodeficiency

the carboxyl terminus of human immunodeficiency virus type 1 gp160 to

Vinkers, M., Guillemont, J., Pasquier, E., Kukla, M., Ludovici, D., Andries, K., de Bethune, M. P., Pauwels, R., Das, K., Clark, A. D., Jr., Frenkel, Y. V., Hughes, S. H., Medaer, B., De Knaep, F., Bohets, H., De Clerck, F., Lampo, A., Williams, P., & Stoffels, P. (2005). In search of a novel anti-HIV drug: multidisciplinary coordination in the discovery of 4-[[4-[[4-[(1E)-2-cyanoethenyl]-2,6-dimethylphenyl]amino]-2 pyrimidinyl]amino]benzonitrile (R278474, rilpivirine). *J Med Chem,* 48(6), 1901-1909. Jensen, M. A., & van 't Wout, A. B. (2003). Predicting HIV-1 coreceptor usage with sequence

& Pommier, Y. (2006). Integration requires a specific interaction of the donor DNA terminal 5'-cytosine with glutamine 148 of the HIV-1 integrase flexible loop. *J Biol*

Schapiro, J. M., & Richman, D. D. (2010). Update of the drug resistance mutations

(2004). Association of a novel human immunodeficiency virus type 1 protease substrate cleft mutation, L23I, with protease inhibitor therapy and in vitro drug

bystander apoptosis is dependent on CCR5 cell surface expression levels as well as

modeled structure of an aptamer-gp120 complex provides insight into the

analysis of an HIV-1 protease I47A mutant resistant to the protease inhibitor

mediated by point mutations in the intracytoplasmic tail of human

Sirivichayakul, S., Soares, M. A., Snoeck, J., Pillay, C., Rudich, H., Rodrigues, R., Holguin, A., Ariyoshi, K., Bouzas, M. B., Cahn, P., Sugiura, W., Soriano, V., Brigido, L. F., Grossman, Z., Morris, L., Vandamme, A. M., Tanuri, A., Phanuphak, P., Weber, J. N., Pillay, D., Harrigan, P. R., Camacho, R., Schapiro, J. M., & Shafer, R. W. (2005). Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration. *PLoS Med,* 2(4), e112.

Point Mutations Associated with HIV-1 Drug

*Virol,* 73(5), 3744-3752.

*AIDS Res Hum Retroviruses,* 22(5), 375-385.

treatment.  *Proc Natl Acad Sci U S A,* 95(23), 13471-13476.

HIV-1 reverse transcriptase. *Mol Cell,* 4(1), 35-43.

cancer cells. *Mol Ther,* 17(1), 153-161.

apoptosis. *J Biol Chem,* 275(2), 1233-1240.

189(5), 837-846.

61(6), 1362-1368.

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 123

Marcelin, A. G., Reynes, J., Yerly, S., Ktorza, N., Segondy, M., Piot, J. C., Delfraissy, J. F.,

Margot, N. A., Lu, B., Cheng, A., & Miller, M. D. (2006). Resistance development over 144

Masquelier, B., Assoumou, K. L., Descamps, D., Bocket, L., Cottalorda, J., Ruffault, A.,

McNicholas, P. M., Mann, P. A., Wojcik, L., Phd, P. Q., Lee, E., McCarthy, M., Shen, J., Black,

phase II study (VICTOR-E1). *J Acquir Immune Defic Syndr,* 56(3), 222-229. Melby, T., Sista, P., DeMasi, R., Kirkland, T., Roberts, N., Salgo, M., Heilek-Snyder, G.,

Menzo, S., Castagna, A., Monachetti, A., Hasson, H., Danise, A., Carini, E., Bagnarelli, P.,

Meyer, P. R., Matsuura, S. E., Mian, A. M., So, A. G., & Scott, W. A. (1999). A mechanism of

Meyer, P. R., Matsuura, S. E., So, A. G., & Scott, W. A. (1998). Unblocking of chain-

Mi, Z., Guo, H., Russell, M. B., Liu, Y., Sullenger, B. A., & Kuo, P. C. (2009). RNA aptamer

Micoli, K. J., Mamaeva, O., Piller, S. C., Barker, J. L., Pan, G., Hunter, E., & McDonald, J. M.

Miller, M. D., Margot, N., Lu, B., Zhong, L., Chen, S. S., Cheng, A., & Wulfsohn, M. (2004).

Mink, M., Mosier, S. M., Janumpalli, S., Davison, D., Jin, L., Melby, T., Sista, P., Erickson, J.,

persistent HIV viraemia under T-20. *Aids,* 18(9), 1340-1342.

Kaiser, L., Perrin, L., Katlama, C., & Calvez, V. (2004c). Characterization of genotypic determinants in HR-1 and HR-2 gp41 domains in individuals with

weeks in treatment-naive patients receiving tenofovir disoproxil fumarate or stavudine with lamivudine and efavirenz in Study 903. *HIV Med,* 7(7), 442-450. Martinez-Picado, J., Savara, A. V., Sutton, L., & D'Aquila, R. T. (1999). Replicative fitness of

protease inhibitor-resistant mutants of human immunodeficiency virus type 1. *J*

Marcelin, A. G., Morand-Joubert, L., Tamalet, C., Charpentier, C., Peytavin, G., Antoun, Z., Brun-Vezinet, F., & Costagliola, D. (2008). Clinically validated mutation scores for HIV-1 resistance to fosamprenavir/ritonavir. *J Antimicrob Chemother,*

T. A., & Strizki, J. M. (2011). Mapping and characterization of vicriviroc resistance mutations from HIV-1 isolated from treatment-experienced subjects enrolled in a

Cammack, N., Matthews, T. J., & Greenberg, M. L. (2006). Characterization of envelope glycoprotein gp41 genotype and phenotypic susceptibility to enfuvirtide at baseline and on treatment in the phase III clinical trials TORO-1 and TORO-2.

Lazzarin, A., & Clementi, M. (2004). Genotype and phenotype patterns of human immunodeficiency virus type 1 resistance to enfuvirtide during long-term

AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant

terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent

blockade of osteopontin inhibits growth and metastasis of MDA-MB231 breast

(2006). Point mutations in the C-terminus of HIV-1 gp160 reduce apoptosis and calmodulin binding without affecting viral replication. *Virology,* 344(2), 468-479. Micoli, K. J., Pan, G., Wu, Y., Williams, J. P., Cook, W. J., & McDonald, J. M. (2000).

Requirement of calmodulin binding by HIV-1 gp160 for enhanced FAS-mediated

Genotypic and phenotypic predictors of the magnitude of response to tenofovir disoproxil fumarate treatment in antiretroviral-experienced patients. *J Infect Dis,*

Lambert, D., Stanfield-Oakley, S. A., Salgo, M., Cammack, N., Matthews, T., &

Liu, Y., Sun, Q. A., Chen, Q., Lee, T. H., Huang, Y., Wetsel, W. C., Michelotti, G. A.,

Low, A. J., Dong, W., Chan, D., Sing, T., Swanstrom, R., Jensen, M., Pillai, S., Good, B., &

Lu, J., Sista, P., Giguel, F., Greenberg, M., & Kuritzkes, D. R. (2004). Relative replicative

Luo, X. M., Lei, M. Y., Feidi, R. A., West, A. P., Jr., Balazs, A. B., Bjorkman, P. J., Yang, L., &

Lupold, S. E., Hicke, B. J., Lin, Y., & Coffey, D. S. (2002). Identification and characterization

Madruga, J. V., Cahn, P., Grinsztejn, B., Haubrich, R., Lalezari, J., Mills, A., Pialoux, G.,

randomised, double-blind, placebo-controlled trial. *Lancet,* 370(9581), 29-38. Malet, I., Delelis, O., Valantin, M. A., Montes, B., Soulie, C., Wirden, M., Tchertanov, L.,

coevolution in protease inhibitor-treated patients. *J Virol,* 72(9), 7632-7637. Marcelin, A. G., Dalban, C., Peytavin, G., Lamotte, C., Agher, R., Delaugerre, C., Wirden, M.,

Marcelin, A. G., Delaugerre, C., Wirden, M., Viegas, P., Simon, A., Katlama, C., & Calvez, V.

Marcelin, A. G., Flandre, P., Pavie, J., Schmidely, N., Wirden, M., Lada, O., Chiche, D.,

resistance to didanosine. *Antimicrob Agents Chemother,* 49(5), 1739-1744.

prostate-specific membrane antigen. *Cancer Res,* 62(14), 4029-4033.

predicting X4 co-receptor usage in clinical isolates. *Aids,* 21(14), F17-24. Lu, J., Deeks, S. G., Hoh, R., Beatty, G., Kuritzkes, B. A., Martin, J. N., & Kuritzkes, D. R.

of a clonal analysis. *J Acquir Immune Defic Syndr,* 43(1), 60-64.

*Neurochem,* 108(1), 147-157.

(T-20). *J Virol,* 78(9), 4628-4637.

*Pathog,* 6(12), e1001225.

4692.

patients. *Antivir Ther,* 12(2), 247-252.

Sullenger, B. A., & Zhang, X. (2009). Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking. *J*

Harrigan, P. R. (2007). Current V3 genotyping algorithms are inadequate for

(2006). Rapid emergence of enfuvirtide resistance in HIV-1-infected patients: results

fitness of human immunodeficiency virus type 1 mutants resistant to enfuvirtide

Baltimore, D. (2011). Dimeric 2G12 as a potent protection against HIV-1. *PLoS*

of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the

Wilkin, T., Peeters, M., Vingerhoets, J., de Smedt, G., Leopold, L., Trefiglio, R., & Woodfall, B. (2007). Efficacy and safety of TMC125 (etravirine) in treatmentexperienced HIV-1-infected patients in DUET-1: 24-week results from a

Peytavin, G., Reynes, J., Mouscadet, J. F., Katlama, C., Calvez, V., & Marcelin, A. G. (2008). Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro. *Antimicrob Agents Chemother,* 52(4), 1351-1358. Mammano, F., Petit, C., & Clavel, F. (1998). Resistance-associated loss of viral fitness in

human immunodeficiency virus type 1: phenotypic analysis of protease and gag

Conan, F., Dantin, S., Katlama, C., Costagliola, D., & Calvez, V. (2004a). Clinically relevant interpretation of genotype and relationship to plasma drug concentrations for resistance to saquinavir-ritonavir in human immunodeficiency virus type 1 protease inhibitor-experienced patients. *Antimicrob Agents Chemother,* 48(12), 4687-

(2004b). Thymidine analogue reverse transcriptase inhibitors resistance mutations profiles and association to other nucleoside reverse transcriptase inhibitors resistance mutations observed in the context of virological failure. *J Med Virol,* 72(1), 162-165. Marcelin, A. G., Flandre, P., de Mendoza, C., Roquebert, B., Peytavin, G., Valer, L., Wirden,

M., Abbas, S., Katlama, C., Soriano, V., & Calvez, V. (2007). Clinical validation of saquinavir/ritonavir genotypic resistance score in protease-inhibitor-experienced

Molina, J. M., & Calvez, V. (2005). Clinically relevant genotype interpretation of

Point Mutations Associated with HIV-1 Drug

8098-8110.

[see comments]. *Lancet,* 349(9060), 1219.

non-V3 epitopes. *J Virol,* 72(9), 7099-7107.

amprenavir cross-resistance. *Aids,* 17(7), 955-961.

*Antimicrob Agents Chemother,* 40(2), 292-297.

regimens failed. *J Infect Dis,* 189(11), 1983-1987.

*Chemother,* 51(4), 1473-1480.

184(8), 983-991.

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 125

O'Brien, T. R., Winkler, C., Dean, M., Nelson, J. A., Carrington, M., Michael, N. L., & White,

Pancera, M., McLellan, J. S., Wu, X., Zhu, J., Changela, A., Schmidt, S. D., Yang, Y., Zhou, T.,

Park, E. J., Vujcic, L. K., Anand, R., Theodore, T. S., & Quinnan, G. V., Jr. (1998). Mutations in

Parkin, N. T., Chappey, C., & Petropoulos, C. J. (2003). Improving lopinavir genotype

Pastore, C., Nedellec, R., Ramos, A., Hartley, O., Miamidian, J. L., Reeves, J. D., & Mosier, D.

Pellegrin, I., Breilh, D., Coureau, G., Boucher, S., Neau, D., Merel, P., Lacoste, D., Fleury, H.,

Pellegrin, I., Breilh, D., Ragnaud, J. M., Boucher, S., Neau, D., Fleury, H., Schrive, M. H.,

pharmacokinetic parameters (Reyaphar study). *Antivir Ther,* 11(4), 421-429. Perno, C. F., Cozzi-Lepri, A., Balotta, C., Forbici, F., Violin, M., Bertoli, A., Facchi, G.,

Perno, C. F., Cozzi-Lepri, A., Forbici, F., Bertoli, A., Violin, M., Stella Mura, M., Cadeo, G.,

Petropoulos, C. J., Parkin, N. T., Limoli, K. L., Lie, Y. S., Wrin, T., Huang, W., Tian, H.,

G. C., 2nd (1997). HIV-1 infection in a man homozygous for CCR5 delta 32 [letter]

Phogat, S., Mascola, J. R., & Kwong, P. D. (2010). Crystal structure of PG16 and chimeric dissection with somatically related PG9: structure-function analysis of two quaternary-specific antibodies that effectively neutralize HIV-1. *J Virol,* 84(16),

both gp120 and gp41 are responsible for the broad neutralization resistance of variant human immunodeficiency virus type 1 MN to antibodies directed at V3 and

algorithm through phenotype correlations: novel mutation patterns and

E. (2007). Conserved changes in envelope function during human immunodeficiency virus type 1 coreceptor switching. *J Virol,* 81(15), 8165-8179. Pastore, C., Nedellec, R., Ramos, A., Pontow, S., Ratner, L., & Mosier, D. E. (2006). Human

immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations. *J Virol,* 80(2), 750-758. Patick, A. K., Mo, H., Markowitz, M., Appelt, K., Wu, B., Musick, L., Kalish, V., Kaldor, S.,

Reich, S., Ho, D., & Webber, S. (1996). Antiviral and resistance studies of AG1343, an orally bioavailable inhibitor of human immunodeficiency virus protease.

Saux, M. C., Pellegrin, J. L., Lazaro, E., Dabis, F., & Thiebaut, R. (2007). Interpretation of genotype and pharmacokinetics for resistance to fosamprenavirritonavir-based regimens in antiretroviral-experienced patients. *Antimicrob Agents*

Saux, M. C., Pellegrin, J. L., Lazaro, E., & Vray, M. (2006). Virological responses to atazanavir-ritonavir-based regimens: resistance-substitutions score and

Pezzotti, P., Cadeo, G., Tositti, G., Pasquinucci, S., Pauluzzi, S., Scalzini, A., Salassa, B., Vincenti, A., Phillips, A. N., Dianzani, F., Appice, A., Angarano, G., Monno, L., Ippolito, G., Moroni, M., & d' Arminio Monforte, A. (2001). Secondary mutations in the protease region of human immunodeficiency virus and virologic failure in drug-naive patients treated with protease inhibitor-based therapy. *J Infect Dis,*

Orani, A., Chirianni, A., De Stefano, C., Balotta, C., & d'Arminio Monforte, A. (2004). Minor mutations in HIV protease at baseline and appearance of primary mutation 90M in patients for whom their first protease-inhibitor antiretroviral

Smith, D., Winslow, G. A., Capon, D. J., & Whitcomb, J. M. (2000). A novel

Greenberg, M. L. (2005). Impact of human immunodeficiency virus type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding and antiviral potency of enfuvirtide in vitro. *J Virol,* 79(19), 12447-12454.

and antiviral potency of enfuvirtide in vitro. *J Virol,* 79(19), 12447-12454. Molina, J. M., Marcelin, A. G., Pavie, J., Heripret, L., De Boever, C. M., Troccaz, M., Leleu, G.,

Moncunill, G., Armand-Ugon, M., Pauls, E., Clotet, B., & Este, J. A. (2008). HIV-1 escape to

Montefiori, D. C., Pantaleo, G., Fink, L. M., Zhou, J. T., Zhou, J. Y., Bilska, M., Miralles, G. D.,

Moog, C., Fleury, H. J., Pellegrin, I., Kirn, A., & Aubertin, A. M. (1997). Autologous and

Moore, J. P., & Sodroski, J. (1996). Antibody cross-competition analysis of the human

Mouscadet, J. F., Arora, R., Andre, J., Lambry, J. C., Delelis, O., Malet, I., Marcelin, A. G.,

Muster, T., Guinea, R., Trkola, A., Purtscher, M., Klima, A., Steindl, F., Palese, P., & Katinger,

Muster, T., Steindl, F., Purtscher, M., Trkola, A., Klima, A., Himmler, G., Ruker, F., &

Nakamura, K. J., Gach, J. S., Jones, L., Semrau, K., Walter, J., Bibollet-Ruche, F., Decker, J. M.,

Nijhuis, M., Schuurman, R., de Jong, D., Erickson, J., Gustchina, E., Albert, J., Schipper, P.,

Nobile, V., Russo, N., Hu, G., & Riordan, J. F. (1998). Inhibition of human angiogenin by

immunodeficiency virus type 1. *J Virol,* 67(11), 6642-6647.

to growth on transformed T cells. *Aids,* 9(Suppl A), S117-136.

840-847.

*Aids,* 22(1), 23-31.

173(1), 60-67.

1863-1872.

4034.

5(3), e9786.

*Defic Syndr,* 38(4), 417-425.

therapy. *Aids,* 13(17), 2349-2359.

*Biochemistry,* 37(19), 6857-6863.

Greenberg, M. L. (2005). Impact of human immunodeficiency virus type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding

& Calvez, V. (2005). Didanosine in HIV-1-infected patients experiencing failure of antiretroviral therapy: a randomized placebo-controlled trial. *J Infect Dis,* 191(6),

CCR5 coreceptor antagonism through selection of CXCR4-using variants in vitro.

& Fauci, A. S. (1996). Neutralizing and infection-enhancing antibody responses to human immunodeficiency virus type 1 in long-term nonprogressors. *J Infect Dis,*

heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals. *J Virol,* 71(5), 3734-3741. Moore, J. P., & Ho, D. D. (1995). HIV-1 neutralization: the consequences of viral adaptation

immunodeficiency virus type 1 gp120 exterior envelope glycoprotein. *J Virol,* 70(3),

Calvez, V., & Tchertanov, L. (2009). HIV-1 IN alternative molecular recognition of DNA induced by raltegravir resistance mutations. *J Mol Recognit,* 22(6), 480-494. Moyle, G. J., DeJesus, E., Cahn, P., Castillo, S. A., Zhao, H., Gordon, D. N., Craig, C., & Scott,

T. R. (2005). Abacavir once or twice daily combined with once-daily lamivudine and efavirenz for the treatment of antiretroviral-naive HIV-infected adults: results of the Ziagen Once Daily in Antiretroviral Combination Study. *J Acquir Immune*

H. (1994). Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS. *J Virol,* 68(6), 4031-

Katinger, H. (1993). A conserved neutralizing epitope on gp41 of human

Heath, L., Decker, W. D., Sinkala, M., Kankasa, C., Thea, D., Mullins, J., Kuhn, L., Zwick, M. B., & Aldrovandi, G. M. (2011). 4E10-resistant HIV-1 isolated from four subjects with rare membrane-proximal external region polymorphisms. *PLoS One,*

Gulnik, S., & Boucher, C. A. (1999). Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal

DNA aptamers: nuclear colocalization of an angiogenin-inhibitor complex.

Point Mutations Associated with HIV-1 Drug

336(3), 569-578.

transcriptases. *J Med Chem,* 50(10), 2301-2309.

*Antimicrob Chemother,* 63(2), 380-388.

surveillance. *J Infect Dis,* 192(3), 456-465.

program to seven non-B subtypes. *Aids,* 20(5), 643-651.

*Nucleic Acids Res,* 31(1), 298-303.

*Agents Chemother,* 48(8), 3122-3126.

*Antivir Ther,* 8(2), 173-182.

*Retroviruses,* 17(12), 1107-1115.

*Proc Natl Acad Sci U S A,* 103(46), 17355-17360.

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 127

Ren, J., Nichols, C. E., Chamberlain, P. P., Weaver, K. L., Short, S. A., & Stammers, D. K.

Ren, J., & Stammers, D. K. (2008). Structural basis for drug resistance mechanisms for nonnucleoside inhibitors of HIV reverse transcriptase. *Virus Res,* 134(1-2), 157-170. Rey, D., Hoen, B., Chavanet, P., Schmitt, M. P., Hoizey, G., Meyer, P., Peytavin, G., Spire, B.,

Rhee, S. Y., Fessel, W. J., Zolopa, A. R., Hurley, L., Liu, T., Taylor, J., Nguyen, D. P., Slome,

Rhee, S. Y., Gonzales, M. J., Kantor, R., Betts, B. J., Ravela, J., & Shafer, R. W. (2003). Human

Rhee, S. Y., Kantor, R., Katzenstein, D. A., Camacho, R., Morris, L., Sirivichayakul, S.,

Rhee, S. Y., Liu, T., Ravela, J., Gonzales, M. J., & Shafer, R. W. (2004). Distribution of human

Rhee, S. Y., Liu, T. F., Holmes, S. P., & Shafer, R. W. (2007). HIV-1 subtype B protease and reverse transcriptase amino acid covariation. *PLoS Comput Biol,* 3(5), e87. Rhee, S. Y., Taylor, J., Wadhera, G., Ben-Hur, A., Brutlag, D. L., & Shafer, R. W. (2006b).

Roge, B. T., Katzenstein, T. L., Obel, N., Nielsen, H., Kirk, O., Pedersen, C., Mathiesen, L.,

Ross, L., Scarsella, A., Raffanti, S., Henry, K., Becker, S., Fisher, R., Liao, Q., Hirani, A.,

Ruckman, J., Green, L. S., Beeson, J., Waugh, S., Gillette, W. L., Henninger, D. D., Claesson-

requiring the exon 7-encoded domain. *J Biol Chem,* 273(32), 20556-20567.

wild-type, Leu100Ile, Lys101Glu, and Tyr188Cys mutant HIV-1 reverse

(2004). Crystal structures of HIV-1 reverse transcriptases mutated at codons 100, 106 and 108 and mechanisms of resistance to non-nucleoside inhibitors. *J Mol Biol,*

Allavena, C., Diemer, M., May, T., Schmit, J. L., Duong, M., Calvez, V., & Lang, J. M. (2009). High rate of early virological failure with the once-daily tenofovir/lamivudine/nevirapine combination in naive HIV-1-infected patients. *J*

S., Klein, D., Horberg, M., Flamm, J., Follansbee, S., Schapiro, J. M., & Shafer, R. W. (2005). HIV-1 Protease and reverse-transcriptase mutations: correlations with antiretroviral therapy in subtype B isolates and implications for drug-resistance

immunodeficiency virus reverse transcriptase and protease sequence database.

Jorgensen, L., Brigido, L. F., Schapiro, J. M., & Shafer, R. W. (2006a). HIV-1 pol mutation frequency by subtype and treatment experience: extension of the HIVseq

immunodeficiency virus type 1 protease and reverse transcriptase mutation patterns in 4,183 persons undergoing genotypic resistance testing. *Antimicrob*

Genotypic predictors of human immunodeficiency virus type 1 drug resistance.

Lundgren, J., & Gerstoft, J. (2003). K65R with and without S68: a new resistance profile in vivo detected in most patients failing abacavir, didanosine and stavudine.

Graham, N., St Clair, M., & Hernandez, J. (2001). Thymidine analog and multinucleoside resistance mutations are associated with decreased phenotypic susceptibility to stavudine in HIV type 1 isolated from zidovudine-naive patients experiencing viremia on stavudine-containing regimens. *AIDS Res Hum*

Welsh, L., & Janjic, N. (1998). 2'-Fluoropyrimidine RNA-based aptamers to the 165 amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions

phenotypic drug susceptibility assay for human immunodeficiency virus type 1. *Antimicrob Agents Chemother,* 44(4), 920-928.

Platt, E. J., Kuhmann, S. E., Rose, P. P., & Kabat, D. (2001). Adaptive mutations in the V3

Platt, E. J., Shea, D. M., Rose, P. P., & Kabat, D. (2005). Variants of human immunodeficiency

Poignard, P., Klasse, P. J., & Sattentau, Q. J. (1996). Antibody neutralization of HIV-1.

Pommier, Y., Johnson, A. A., & Marchand, C. (2005). Integrase inhibitors to treat HIV/AIDS.

Posner, M. R., Hideshima, T., Cannon, T., Mukherjee, M., Mayer, K. H., & Byrn, R. A. (1991).

Reeves, J. D., Lee, F. H., Miamidian, J. L., Jabara, C. B., Juntilla, M. M., & Doms, R. W. (2005).

Reitz, M. S., Jr., Wilson, C., Naugle, C., Gallo, R. C., & Robert-Guroff, M. (1988). Generation

Ren, J., Chamberlain, P. P., Stamp, A., Short, S. A., Weaver, K. L., Romines, K. R., Hazen, R.,

Ren, J., Esnouf, R., Garman, E., Somers, D., Ross, C., Kirby, I., Keeling, J., Darby, G., Jones,

Ren, J., Esnouf, R., Hopkins, A., Ross, C., Jones, Y., Stammers, D., & Stuart, D. (1995b). The

Ren, J., Nichols, C., Bird, L., Chamberlain, P., Weaver, K., Short, S., Stuart, D. I., & Stammers,

Ren, J., Nichols, C. E., Chamberlain, P. P., Weaver, K. L., Short, S. A., Chan, J. H., Kleim, J. P.,

generation non-nucleoside inhibitors. *J Mol Biol,* 312(4), 795-805.

binding to cells, and neutralizes infection. *J Immunol,* 146(12), 4325-4332. Rana, S., Besson, G., Cook, D. G., Rucker, J., Smyth, R. J., Yi, Y., Turner, J. D., Guo, H. H., Du,

resulting from the delta ccr5 mutation. *J Virol,* 71(4), 3219-3227.

*Antimicrob Agents Chemother,* 44(4), 920-928.

*Virol,* 75(24), 12266-12278.

*Immunol Today,* 17(5), 239-246.

*Nat Rev Drug Discov,* 4(3), 236-248.

in the envelope gene. *Cell,* 54(1), 57-63.

inhibitor complexes. *Nat Struct Biol,* 2(4), 293-302.

inhibitor design. *Structure,* 3(9), 915-926.

*Med Chem,* 51(16), 5000-5008.

79(7), 4357-4368.

79(8), 4991-4999.

phenotypic drug susceptibility assay for human immunodeficiency virus type 1.

loop of gp120 enhance fusogenicity of human immunodeficiency virus type 1 and enable use of a CCR5 coreceptor that lacks the amino-terminal sulfated region. *J*

virus type 1 that efficiently use CCR5 lacking the tyrosine-sulfated amino terminus have adaptive mutations in gp120, including loss of a functional N-glycan. *J Virol,*

An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits virus

J. G., Peiper, S. C., Lavi, E., Samson, M., Libert, F., Liesnard, C., Vassart, G., Doms, R. W., Parmentier, M., & Collman, R. G. (1997). Role of CCR5 in infection of primary macrophages and lymphocytes by macrophage-tropic strains of human immunodeficiency virus: resistance to patient-derived and prototype isolates

Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization. *J Virol,*

of a neutralization-resistant variant of HIV-1 is due to selection for a point mutation

Freeman, A., Ferris, R. G., Andrews, C. W., Boone, L., Chan, J. H., & Stammers, D. K. (2008). Structural basis for the improved drug resistance profile of new generation benzophenone non-nucleoside HIV-1 reverse transcriptase inhibitors. *J*

Y., Stuart, D., & et al. (1995a). High resolution structures of HIV-1 RT from four RT-

structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for

D. K. (2001). Structural mechanisms of drug resistance for mutations at codons 181 and 188 in HIV-1 reverse transcriptase and the improved resilience of second

& Stammers, D. K. (2007). Relationship of potency and resilience to drug resistant mutations for GW420867X revealed by crystal structures of inhibitor complexes for wild-type, Leu100Ile, Lys101Glu, and Tyr188Cys mutant HIV-1 reverse transcriptases. *J Med Chem,* 50(10), 2301-2309.

Point Mutations Associated with HIV-1 Drug

1097-1106.

42-53.

*Acquir Immune Defic Syndr,* 23(3), 221-226.

susceptibility in vitro. *J Clin Virol,* 36(4), 249-257.

*J Acquir Immune Defic Syndr,* 46(2), 174-180.

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 129

Sing, T., Low, A. J., Beerenwinkel, N., Sander, O., Cheung, P. K., Domingues, F. S., Buch, J.,

Sista, P. R., Melby, T., Davison, D., Jin, L., Mosier, S., Mink, M., Nelson, E. L., DeMasi, R.,

treatment of HIV infection: 48-week results of ALERT. *AIDS Res Ther,* 5, 5. Sosa, N., Hill-Zabala, C., Dejesus, E., Herrera, G., Florance, A., Watson, M., Vavro, C., &

Sugiura, W., Matsuda, Z., Yokomaku, Y., Hertogs, K., Larder, B., Oishi, T., Okano, A.,

immunodeficiency virus type 1. *Antimicrob Agents Chemother,* 46(3), 708-715. Svarovskaia, E. S., Margot, N. A., Bae, A. S., Waters, J. M., Goodman, D., Zhong, L., Borroto-

Svicher, V., Balestra, E., Cento, V., Sarmati, L., Dori, L., Vandenbroucke, I., D'Arrigo, R.,

Svicher, V., Ceccherini-Silberstein, F., Erba, F., Santoro, M., Gori, C., Bellocchi, M. C.,

Theodorou, I., Meyer, L., Magierowska, M., Katlama, C., & Rouzioux, C. (1997). HIV-1

Tilton, J. C., Wilen, C. B., Didigu, C. A., Sinha, R., Harrison, J. E., Agrawal-Gamse, C.,

[letter] [see comments]. *Lancet,* 349(9060), 1219-1220.

experienced patients: clinical and genotypic predictors of virologic response. *J*

Daumer, M., Kaiser, R., Lengauer, T., & Harrigan, P. R. (2007). Predicting HIV coreceptor usage on the basis of genetic and clinical covariates. *Antivir Ther,* 12(7),

Cammack, N., Salgo, M. P., Matthews, T. J., & Greenberg, M. L. (2004). Characterization of determinants of genotypic and phenotypic resistance to enfuvirtide in baseline and on-treatment HIV-1 isolates. *Aids,* 18(13), 1787-1794. Smith, K. Y., Weinberg, W. G., Dejesus, E., Fischl, M. A., Liao, Q., Ross, L. L., Pakes, G. E.,

Pappa, K. A., & Lancaster, C. T. (2008). Fosamprenavir or atazanavir once daily boosted with ritonavir 100 mg, plus tenofovir/emtricitabine, for the initial

Shaefer, M. (2005). Abacavir and lamivudine fixed-dose combination tablet once daily compared with abacavir and lamivudine twice daily in HIV-infected patients over 48 weeks (ESS30008, SEAL). *J Acquir Immune Defic Syndr,* 40(4), 422-427. Su, C., Melby, T., DeMasi, R., Ravindran, P., & Heilek-Snyder, G. (2006). Genotypic changes

in human immunodeficiency virus type 1 envelope glycoproteins on treatment with the fusion inhibitor enfuvirtide and their influence on changes in drug

Shiino, T., Tatsumi, M., Matsuda, M., Abumi, H., Takata, N., Shirahata, S., Yamada, K., Yoshikura, H., & Nagai, Y. (2002). Interference between D30N and L90M in selection and development of protease inhibitor-resistant human

Esoda, K., & Miller, M. D. (2007). Low-level K65R mutation in HIV-1 reverse transcriptase of treatment-experienced patients exposed to abacavir or didanosine.

Buonomini, A. R., Van Marck, H., Surdo, M., Saccomandi, P., Mostmans, W., Aerssens, J., Aquaro, S., Stuyver, L. J., Andreoni, M., Ceccherini-Silberstein, F., & Perno, C. F. (2011). HIV-1 dual/mixed tropic isolates show different genetic and phenotypic characteristics and response to maraviroc in vitro. *Antiviral Res,* 90(1),

Giannella, S., Trotta, M. P., Monforte, A., Antinori, A., & Perno, C. F. (2005). Novel human immunodeficiency virus type 1 protease mutations potentially involved in resistance to protease inhibitors. *Antimicrob Agents Chemother,* 49(5), 2015-2025. Thali, M., Moore, J. P., Furman, C., Charles, M., Ho, D. D., Robinson, J., & Sodroski, J. (1993).

 *J Virol,* 67(7), 3978-3988.

infection in an individual homozygous for CCR5 delta 32. Seroco Study Group

Henning, E. A., Bushman, F. D., Martin, J. N., Deeks, S. G., & Doms, R. W. (2011). A

Ruprecht, C. R., Krarup, A., Reynell, L., Mann, A. M., Brandenberg, O. F., Berlinger, L.,

Rusconi, C. P., Roberts, J. D., Pitoc, G. A., Nimjee, S. M., White, R. R., Quick, G., Jr., Scardino,

Rusconi, C. P., Scardino, E., Layzer, J., Pitoc, G. A., Ortel, T. L., Monroe, D., & Sullenger, B.

Samson, M., Libert, F., Doranz, B. J., Rucker, J., Liesnard, C., Farber, C. M., Saragosti, S.,

Sarafianos, S. G., Das, K., Clark, A. D., Jr., Ding, J., Boyer, P. L., Hughes, S. H., & Arnold, E.

Sarafianos, S. G., Das, K., Hughes, S. H., & Arnold, E. (2004). Taking aim at a moving target:

Sarafianos, S. G., Marchand, B., Das, K., Himmel, D. M., Parniak, M. A., Hughes, S. H., &

Seclen, E., Gonzalez Mdel, M., Lapaz, M., Rodriguez, C., del Romero, J., Aguilera, A., de

Shafer, R. W., & Schapiro, J. M. (2008). HIV-1 drug resistance mutations: an updated framework for the second decade of HAART. *AIDS Rev,* 10(2), 67-84. Shimura, K., Kodama, E., Sakagami, Y., Matsuzaki, Y., Watanabe, W., Yamataka, K.,

Shulman, N. S., Bosch, R. J., Mellors, J. W., Albrecht, M. A., & Katzenstein, D. A. (2004). Genetic correlates of efavirenz hypersusceptibility. *Aids,* 18(13), 1781-1785. Shulman, N. S., Zolopa, A. R., Passaro, D. J., Murlidharan, U., Israelski, D. M., Brosgart, C.

mechanisms of polymerization and inhibition. *J Mol Biol,* 385(3), 693-713. Sattentau, Q. J., & Moore, J. P. (1995). Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer. *J Exp Med,* 182(1), 185-196. Scanlan, C. N., Pantophlet, R., Wormald, M. R., Saphire, E. O., Calarese, D., Stanfield, R.,

anticoagulant aptamer in vivo. *Nat Biotechnol,* 22(11), 1423-1428.

[see comments]. *Nature,* 382(6593), 722-725.

*Med,* 208(3), 439-454.

419(6902), 90-94.

10032.

*Struct Biol,* 14(6), 716-730.

*Biol,* 535, 205-218.

82(2), 764-774.

*Chemother,* 65(12), 2502-2504.

Abela, I. A., Regoes, R. R., Gunthard, H. F., Rusert, P., & Trkola, A. (2011). MPERspecific antibodies induce gp120 shedding and irreversibly neutralize HIV-1. *J Exp*

E., Fay, W. P., & Sullenger, B. A. (2004). Antidote-mediated control of an

A. (2002). RNA aptamers as reversible antagonists of coagulation factor IXa. *Nature,*

Lapoumeroulie, C., Cognaux, J., Forceille, C., Muyldermans, G., Verhofstede, C., Burtonboy, G., Georges, M., Imai, T., Rana, S., Yi, Y., Smyth, R. J., Collman, R. G., Doms, R. W., Vassart, G., & Parmentier, M. (1996). Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene

(1999). Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids. *Proc Natl Acad Sci U S A,* 96(18), 10027-

designing drugs to inhibit drug-resistant HIV-1 reverse transcriptases. *Curr Opin*

Arnold, E. (2009). Structure and function of HIV-1 reverse transcriptase: molecular

Wilson, I. A., Katinger, H., Dwek, R. A., Burton, D. R., & Rudd, P. M. (2003). The carbohydrate epitope of the neutralizing anti-HIV-1 antibody 2G12. *Adv Exp Med*

Mendoza, C., Soriano, V., & Poveda, E. (2011). Primary resistance to maraviroc in a large set of R5-V3 viral sequences from HIV-1-infected patients. *J Antimicrob*

Watanabe, Y., Ohata, Y., Doi, S., Sato, M., Kano, M., Ikeda, S., & Matsuoka, M. (2008). Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor elvitegravir (JTK-303/GS-9137). *J Virol,*

L., Miller, M. D., Van Doren, S., Shafer, R. W., & Katzenstein, D. A. (2000). Efavirenz- and adefovir dipivoxil-based salvage therapy in highly treatmentexperienced patients: clinical and genotypic predictors of virologic response. *J Acquir Immune Defic Syndr,* 23(3), 221-226.

Point Mutations Associated with HIV-1 Drug

*Natl Acad Sci U S A,* 91(21), 9770-9774.

*Immune Defic Syndr,* 48(1), 26-34.

*Dis,* 188(4), 537-540.

*Science,* 329(5993), 856-861.

*Chemother,* 49(3), 1113-1119.

Resistance, Evasion of the Immune Response and AIDS Pathogenesis 131

immunodeficiency virus type 1 clinical isolates. *J Infect Dis,* 188(7), 992-1000. White, R. R., Shan, S., Rusconi, C. P., Shetty, G., Dewhirst, M. W., Kontos, C. D., & Sullenger,

Winters, B., Montaner, J., Harrigan, P. R., Gazzard, B., Pozniak, A., Miller, M. D., Emery, S.,

Winters, M. A., Bosch, R. J., Albrecht, M. A., & Katzenstein, D. A. (2003). Clinical impact of

Winters, M. A., Shafer, R. W., Jellinger, R. A., Mamtora, G., Gingeras, T., & Merigan, T. C.

monotherapy for 1 to 2 years. *Antimicrob Agents Chemother,* 41(4), 757-762. Wu, L., Gerard, N. P., Wyatt, R., Choe, H., Parolin, C., Ruffing, N., Borsetti, A., Cardoso, A.

Wu, T. D., Schiffer, C. A., Gonzales, M. J., Taylor, J., Kantor, R., Chou, S., Israelski, D.,

Wu, X., Yang, Z. Y., Li, Y., Hogerkorp, C. M., Schief, W. R., Seaman, M. S., Zhou, T.,

Wyatt, R., Kwong, P. D., Desjardins, E., Sweet, R. W., Robinson, J., Hendrickson, W. A., &

Xu, L., Pozniak, A., Wildfire, A., Stanfield-Oakley, S. A., Mosier, S. M., Ratcliffe, D.,

CCR-5 [see comments]. *Nature,* 384(6605), 179-183.

protease inhibitor treatments. *J Virol,* 77(8), 4836-4847.

glycoprotein [see comments]. *Nature,* 393(6686), 705-711.

susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry. *J Virol,* 81(5), 2359-2371. Whitcomb, J. M., Parkin, N. T., Chappey, C., Hellmann, N. S., & Petropoulos, C. J. (2003).

Broad nucleoside reverse-transcriptase inhibitor cross-resistance in human

B. A. (2003). Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2. *Proc Natl Acad Sci U S A,* 100(9), 5028-5033. Wild, C. T., Shugars, D. C., Greenwell, T. K., McDanal, C. B., & Matthews, T. J. (1994).

Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. *Proc*

van Leth, F., Robinson, P., Baxter, J. D., Perez-Elias, M., Castor, D., Hammer, S., Rinehart, A., Vermeiren, H., Van Craenenbroeck, E., & Bacheler, L. (2008). Determination of clinically relevant cutoffs for HIV-1 phenotypic resistance estimates through a combined analysis of clinical trial and cohort data. *J Acquir*

the M184V mutation on switching to didanosine or maintaining lamivudine treatment in nucleoside reverse-transcriptase inhibitor-experienced patients. *J Infect*

(1997). Human immunodeficiency virus type 1 reverse transcriptase genotype and drug susceptibility changes in infected individuals receiving dideoxyinosine

A., Desjardin, E., Newman, W., Gerard, C., & Sodroski, J. (1996). CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor

Zolopa, A. R., Fessel, W. J., & Shafer, R. W. (2003). Mutation patterns and structural correlates in human immunodeficiency virus type 1 protease following different

Schmidt, S. D., Wu, L., Xu, L., Longo, N. S., McKee, K., O'Dell, S., Louder, M. K., Wycuff, D. L., Feng, Y., Nason, M., Doria-Rose, N., Connors, M., Kwong, P. D., Roederer, M., Wyatt, R. T., Nabel, G. J., & Mascola, J. R. (2010). Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1.

Sodroski, J. G. (1998). The antigenic structure of the HIV gp120 envelope

Workman, J., Joall, A., Myers, R., Smit, E., Cane, P. A., Greenberg, M. L., & Pillay, D. (2005). Emergence and evolution of enfuvirtide resistance following long-term therapy involves heptad repeat 2 mutations within gp41. *Antimicrob Agents*

maraviroc-resistant HIV-1 with narrow cross-resistance to other CCR5 antagonists depends on both N-terminal and extracellular loop domains of drug-bound CCR5. *J Virol,* 84(20), 10863-10876.

Trkola, A., Dragic, T., Arthos, J., Binley, J. M., Olson, W. C., Allaway, G. P., Cheng-Mayer,

Trkola, A., Purtscher, M., Muster, T., Ballaun, C., Buchacher, A., Sullivan, N., Srinivasan, K.,

van Maarseveen, N. M., de Jong, D., Boucher, C. A., & Nijhuis, M. (2006). An increase in

Vermeiren, H., Van Craenenbroeck, E., Alen, P., Bacheler, L., Picchio, G., & Lecocq, P. (2007).

Vinores, S. A. (2003). Technology evaluation: pegaptanib, Eyetech/Pfizer. *Curr Opin Mol*

Vora, S., Marcelin, A. G., Gunthard, H. F., Flandre, P., Hirsch, H. H., Masquelier, B.,

Vray, M., Meynard, J. L., Dalban, C., Morand-Joubert, L., Clavel, F., Brun-Vezinet, F.,

standard of care (Narval trial, ANRS 088). *Antivir Ther,* 8(5), 427-434. Walker, L. M., Phogat, S. K., Chan-Hui, P. Y., Wagner, D., Phung, P., Goss, J. L., Wrin, T.,

human immunodeficiency virus type 1. *J Virol,* 70(2), 1100-1108.

linear regression modeling. *J Virol Methods,* 145(1), 47-55.

experienced patients. *Aids,* 20(1), 35-40.

neutralizing antibodies. *PLoS One,* 6(3), e18233.

entire HIV-1 RNA genome. *Nature,* 460(7256), 711-716.

*J Virol,* 84(20), 10863-10876.

*Nature,* 384(6605), 184-187.

*Syndr,* 42(2), 162-168.

*Ther,* 5(6), 673-679.

maraviroc-resistant HIV-1 with narrow cross-resistance to other CCR5 antagonists depends on both N-terminal and extracellular loop domains of drug-bound CCR5.

C., Robinson, J., Maddon, P. J., & Moore, J. P. (1996a). CD4-dependent, antibodysensitive interactions between HIV-1 and its co- receptor CCR-5 [see comments].

Sodroski, J., Moore, J. P., & Katinger, H. (1996b). Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of

viral replicative capacity drives the evolution of protease inhibitor-resistant human immunodeficiency virus type 1 in the absence of drugs. *J Acquir Immune Defic*

Prediction of HIV-1 drug susceptibility phenotype from the viral genotype using

Zinkernagel, A., Peytavin, G., Calvez, V., Perrin, L., & Yerly, S. (2006). Clinical validation of atazanavir/ritonavir genotypic resistance score in protease inhibitor-

Peytavin, G., Costagliola, D., & Girard, P. M. (2003). Predictors of the virological response to a change in the antiretroviral treatment regimen in HIV-1-infected patients enrolled in a randomized trial comparing genotyping, phenotyping and

Simek, M. D., Fling, S., Mitcham, J. L., Lehrman, J. K., Priddy, F. H., Olsen, O. A., Frey, S. M., Hammond, P. W., Kaminsky, S., Zamb, T., Moyle, M., Koff, W. C., Poignard, P., & Burton, D. R. (2009). Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. *Science,* 326(5950), 285-289. Walmsley, S. L., Kelly, D. V., Tseng, A. L., Humar, A., & Harrigan, P. R. (2001). Non-

nucleoside reverse transcriptase inhibitor failure impairs HIV-RNA responses to

with exposed membrane-proximal external region inducing broad HIV-1

(1999). CCR5 HIV-1 coreceptor activity. Role of cooperativity between residues in Nterminal extracellular and intracellular domains. *J Biol Chem,* 274(40), 28413-28419. Watts, J. M., Dang, K. K., Gorelick, R. J., Leonard, C. W., Bess, J. W., Jr., Swanstrom, R.,

Burch, C. L., & Weeks, K. M. (2009). Architecture and secondary structure of an

Ciaramella, G., & Perros, M. (2007). Reduced maximal inhibition in phenotypic

efavirenz-containing salvage antiretroviral therapy. *Aids,* 15(12), 1581-1584. Wang, J., Tong, P., Lu, L., Zhou, L., Xu, L., Jiang, S., & Chen, Y. H. (2011). HIV-1 gp41 core

Wang, Z., Lee, B., Murray, J. L., Bonneau, F., Sun, Y., Schweickart, V., Zhang, T., & Peiper, S. C.

Westby, M., Smith-Burchnell, C., Mori, J., Lewis, M., Mosley, M., Stockdale, M., Dorr, P.,

susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry. *J Virol,* 81(5), 2359-2371.

**6**

**Recombination and Point**

Abid Nabil Ben Salem1, Rouis Zyed1, Buesa Javier2 and Aouni Mahjoub1 *1Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives LR99-ES27, Faculté de Pharmacie, Université de Monastir, 2Dept. de Microbiología, Facultad de Medicina y*

*Avda. Blasco Ibañez, Valencia,*

*1Tunisia 2Spain*

*Hospital Clínico Universitario, Universidad de Valencia,*

**Mutations in Type G Rotavirus Strains:**

**The Challenges of Vaccine Development**

Active immunity refers to the process of exposing the body to an antigen to generate an adaptive immune response: the response takes days/weeks to develop but may be long lasting—even lifelong. Wild infection with pathogenic agents (eg. Hepatitis A Virus) and subsequent recovery gives rise to a natural active immune response usually leading to lifelong protection. In addition, some infections can be prevented by immunization with

The term "vaccine" is derived from the Latin word "vaccinus" which means "pertaining to cows" – a reection on Jenner's pioneering studies using cowpox vaccinia virus to prevent human smallpox (variola) as discussed previously (Stefan, 2005; Dunn, 1996). Vaccines take advantage of using relatively harmless foreign agents to evoke protective immunity for protection against several important pathogens. Vaccine development has its early roots in the work of Edward Jenner and Louis Pasteur, who discovered how to protect people from

All vaccines contain other substances (termed excipients) that are present because they improve the immune response (an adjuvant), are necessary for ensuring stability of the product (stabilizers and preservatives), are the vehicle for delivering vaccine (carrier) or are a residual of the manufacturing process (for example antibiotics or cell culture components). Nowadays, many types of vaccines have been proposed and used in vaccine development: Live whole virus vaccines, Killed whole virus vaccines, Subunit vaccines (purified or recombinant viral antigen), Toxoid vaccines, Synthetic vaccines, and DNA vaccines. We will

smallpox and developed a vaccine to protect from rabies, respectively.

discuss these vaccine types one-by-one.

**1. Introduction**

vaccines.

### **Recombination and Point Mutations in Type G Rotavirus Strains: The Challenges of Vaccine Development**

Abid Nabil Ben Salem1, Rouis Zyed1, Buesa Javier2 and Aouni Mahjoub1

*1Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives LR99-ES27, Faculté de Pharmacie, Université de Monastir, 2Dept. de Microbiología, Facultad de Medicina y Hospital Clínico Universitario, Universidad de Valencia, Avda. Blasco Ibañez, Valencia, 1Tunisia 2Spain*

#### **1. Introduction**

132 Point Mutation

Yahi, N., Tamalet, C., Tourres, C., Tivoli, N., Ariasi, F., Volot, F., Gastaut, J. A., Gallais, H.,

Yahi, N., Tamalet, C., Tourres, C., Tivoli, N., & Fantini, J. (2000). Mutation L210W of HIV-1

Yuan, Y., Maeda, Y., Terasawa, H., Monde, K., Harada, S., & Yusa, K. (2011). A combination

Zhou, T., Georgiev, I., Wu, X., Yang, Z. Y., Dai, K., Finzi, A., Kwon, Y. D., Scheid, J. F., Shi,

Zimmerman, P. A., Buckler-White, A., Alkhatib, G., Spalding, T., Kubofcik, J., Combadiere,

Zolopa, A. R., Shafer, R. W., Warford, A., Montoya, J. G., Hsu, P., Katzenstein, D., Merigan,

Zwick, M. B., Jensen, R., Church, S., Wang, M., Stiegler, G., Kunert, R., Katinger, H., & Burton,

region of glycoprotein gp41 to neutralize HIV-1. *J Virol,* 79(2), 1252-1261. Zwick, M. B., Labrijn, A. F., Wang, M., Spenlehauer, C., Saphire, E. O., Binley, J. M., Moore, J.

neutralization of HIV-1 by antibody VRC01. *Science,* 329(5993), 811-817. Zhu, D. M., Shi, J., Liu, S., Liu, Y., & Zheng, D. (2011). HIV infection enhances TRAIL-

and generation of reactive oxygen species. *PLoS One,* 6(4), e18291.

4099-4106.

transcriptase. *J Biomed Sci,* 7(6), 507-513.

quantified risk. *Mol Med,* 3(1), 23-36.

therapy had failed. *Ann Intern Med,* 131(11), 813-821.

resistance to maraviroc. *Virology,* 413(2), 293-299.

Moreau, J., & Fantini, J. (1999). Mutation patterns of the reverse transcriptase and protease genes in human immunodeficiency virus type 1-infected patients undergoing combination therapy: survey of 787 sequences. *J Clin Microbiol,* 37(12),

reverse transcriptase in patients receiving combination therapy. Incidence, association with other mutations, and effects on the structure of mutated reverse

of polymorphic mutations in V3 loop of HIV-1 gp120 can confer noncompetitive

W., Xu, L., Yang, Y., Zhu, J., Nussenzweig, M. C., Sodroski, J., Shapiro, L., Nabel, G. J., Mascola, J. R., & Kwong, P. D. (2011). Structural basis for broad and potent

induced cell death in macrophage by down-regulating decoy receptor expression

C., Weissman, D., Cohen, O., Rubbert, A., Lam, G., Vaccarezza, M., Kennedy, P. E., Kumaraswami, V., Giorgi, J. V., Detels, R., Hunter, J., Chopek, M., Berger, E. A., Fauci, A. S., Nutman, T. B., & Murphy, P. M. (1997). Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and

T. C., & Efron, B. (1999). HIV-1 genotypic resistance patterns predict response to saquinavir-ritonavir therapy in patients in whom previous protease inhibitor

D. R. (2005). Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external

P., Stiegler, G., Katinger, H., Burton, D. R., & Parren, P. W. (2001). Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. *J Virol,* 75(22), 10892-10905.

Active immunity refers to the process of exposing the body to an antigen to generate an adaptive immune response: the response takes days/weeks to develop but may be long lasting—even lifelong. Wild infection with pathogenic agents (eg. Hepatitis A Virus) and subsequent recovery gives rise to a natural active immune response usually leading to lifelong protection. In addition, some infections can be prevented by immunization with vaccines.

The term "vaccine" is derived from the Latin word "vaccinus" which means "pertaining to cows" – a reection on Jenner's pioneering studies using cowpox vaccinia virus to prevent human smallpox (variola) as discussed previously (Stefan, 2005; Dunn, 1996). Vaccines take advantage of using relatively harmless foreign agents to evoke protective immunity for protection against several important pathogens. Vaccine development has its early roots in the work of Edward Jenner and Louis Pasteur, who discovered how to protect people from smallpox and developed a vaccine to protect from rabies, respectively.

All vaccines contain other substances (termed excipients) that are present because they improve the immune response (an adjuvant), are necessary for ensuring stability of the product (stabilizers and preservatives), are the vehicle for delivering vaccine (carrier) or are a residual of the manufacturing process (for example antibiotics or cell culture components).

Nowadays, many types of vaccines have been proposed and used in vaccine development: Live whole virus vaccines, Killed whole virus vaccines, Subunit vaccines (purified or recombinant viral antigen), Toxoid vaccines, Synthetic vaccines, and DNA vaccines. We will discuss these vaccine types one-by-one.

Recombination and Point Mutations in

**1.4 Toxoid vaccines**

are used out in the community.

**1.5 Synthetic peptides**

**1.6 DNA vaccines**

Type G Rotavirus Strains: The Challenges of Vaccine Development 135

Virus proteins have been expressed in bacteria, yeast, mammalian cells, and viruses. *E. Coli* cells were first to be used for this purpose but the expressed proteins were not glycosylated, which was a major drawback since many of the immunogenic proteins of viruses such as the envelope glycoproteins, were glycosylated. Nevertheless, in many instances, it was demonstrated that the non-glycosylated protein backbone was just as immunogenic. Recombinant hepatitis B vaccine is the only recombinant vaccine licensed at present (Yap et al., 1992). An alternative application of recombinant DNA technology is the production of hybrid virus vaccines. The best known example is vaccinia (Smith et al., 1983). The recombinant virus vaccine can then multiply in infected cells and produce the antigens of a wide range of viruses. The genes of several viruses can be inserted, so the potential exists for producing polyvalent live vaccines (Hauser et al., 1988; Hilleman, 1987).

Certain pathogens cause disease by secreting an exotoxin: these include tetanus, diphtheria, botulism and cholera. For these bacteria that secrete toxins, or harmful chemicals, a toxoid vaccine might be the answer. These vaccines are used when a bacterial toxin is the main cause of illness. Scientists have found that they can inactivate toxins by treating them with formalin, a solution of formaldehyde and sterilized water. Such "detoxified" toxins, called toxoids, are safe for use in vaccines. When the immune system receives a vaccine containing a harmless toxoid, it learns how to fight off the natural toxin. The immune system produces antibodies that lock onto and block the toxin. Vaccines against diphtheria and tetanus are examples of toxoid vaccines (Bizzini et al., 1970; Alouf, 1987). These vaccines are: safe because they cannot cause the disease they prevent as there is no possibility of reversion to virulence; the vaccine antigens are not actively multiplying, they cannot spread to unimmunized individuals; they are usually stable and long lasting as they are less susceptible to changes in temperature, humidity and light which can result when vaccines

The development of synthetic peptides that might be useful as vaccines depends on the identification of immunogenic sites (Milich, 1990; Hans et al., 2006; Dorothea, 1993; Jonathan, 1987). Synthetic peptide vaccines have been successfully developed for the immunoprophylaxis of infection with foot-and-mouth disease virus (Bittle et al., 1982; Brown, 1990), type A influenza virus (Muller et al., 1982), and poliovirus (Emini et al., 1983). Synthetic peptide vaccines would have many advantages. Their antigens are precisely defined and free from unnecessary components which may be associated with side effects. They are stable and relatively cheap to manufacture. Changes due to natural variation of the virus can be readily accommodated, which would be a great advantage for unstable viruses.

The demonstration by Wolff and colleagues in 1990 (Wolff et al., 1990) that protein could be expressed following direct inoculation of plasmid DNA into muscle tissue unveiled an

#### **1.1 Live, attenuated virus vaccines**

They are prepared from attenuated strains that are almost or completely devoid of pathogenicity but are capable of inducing a protective immune response. They multiply in the human host and provide continuous antigenic stimulation over a period of time. Primary vaccine failures are uncommon and are usually the result of inadequate storage or administration. Another possibility is interference by related viruses as is suspected in the case of oral polio vaccine in developing countries (Giammanco et al., 1988; Drozdov & Shirman, 1961; Katz & Plotkin, 1968). Several methods have been used to attenuate viruses for vaccine production such as the use of a related virus from another animal (cowpox to prevent smallpox), the administration of pathogenic or partially attenuated virus by an unnatural route, passage of the virus in an "unnatural host" or host cell (e.g. the 17D strain of yellow fever was developed by passage in mice and then in chick embryos (Norrby, 2007) and Polioviruses were passaged in monkey kidney cells (Chezzi et al., 1998) and measles in chick embryo fibroblasts (Katz, 1958), and the development of temperature sensitive mutants (Pringle, 1996).

#### **1.2 Killed/Inactivated vaccines**

The term killed generally refers to bacterial vaccines, whereas inactivated relates to viral vaccines (Levine et al., 1997). They were the easiest preparations to use. The preparation was simply inactivated. For viruses, the outer virion coat should be left intact but the replicative function should be destroyed. To be effective, non-replicating virus vaccines must contain much more antigen than live vaccines that are able to replicate in the host. Preparation of killed vaccines may take the route of heat or chemicals (Turner et al., 1970). The chemicals used include formaldehyde or beta-propiolactone (Lo Grippo, 1960; Gard, 1960). The traditional agent for inactivation of the virus is formalin (Weil & Gall, 1940; Kim & Sharp, 1967). Excessive treatment with this detergent can destroy immunogenicity whereas insufficient treatment can leave infectious virus capable of causing disease. Soon after the introduction of inactivated polio vaccine, there was an outbreak of paralytic poliomyelitis in the USA due to the distribution of inadequately inactivated polio vaccine (Prevots et al., 1996). This incident led to a review of the formalin inactivation procedure and other inactivating agents are available, such as beta-propiolactone. Another problem was that SV40 was occasionally found as a contaminant and there were fears of the potential oncogenic nature of the virus (Tam et al., 2004).

#### **1.3 Subunit vaccines**

Originally, non-replicating vaccines were derived from crude preparations of virus from animal tissues. As the technology for growing viruses to high titres in cell cultures advanced, it became practical to purify virus and viral antigens. It is now possible to identify the peptide sites encompassing the major antigenic sites of viral antigens, from which highly purified subunit vaccines can be produced. Increasing purification may lead to loss of immunogenicity, and this may necessitate coupling to an immunogenic carrier protein or adjuvant, such as an aluminum salt. Examples of purified subunit vaccines include the HA vaccines for influenza A and B (Bachmayer et al., 1976), and HBsAg derived from the plasma of carriers (Vyas et al., 1984). Subunit vaccines can be further subdivided into those where the antigen is produced using recombinant DNA technology and those based on normal bacteriological growth processes.

Virus proteins have been expressed in bacteria, yeast, mammalian cells, and viruses. *E. Coli* cells were first to be used for this purpose but the expressed proteins were not glycosylated, which was a major drawback since many of the immunogenic proteins of viruses such as the envelope glycoproteins, were glycosylated. Nevertheless, in many instances, it was demonstrated that the non-glycosylated protein backbone was just as immunogenic. Recombinant hepatitis B vaccine is the only recombinant vaccine licensed at present (Yap et al., 1992). An alternative application of recombinant DNA technology is the production of hybrid virus vaccines. The best known example is vaccinia (Smith et al., 1983). The recombinant virus vaccine can then multiply in infected cells and produce the antigens of a wide range of viruses. The genes of several viruses can be inserted, so the potential exists for producing polyvalent live vaccines (Hauser et al., 1988; Hilleman, 1987). HBsAg, rabies, HSV and other viruses have been expressed in vaccinia (Mackett et al., 1985; Panicali et al., 1983; Paoletti et al., 1984; Perkus et al., 1985; Rice et al., 1985; Smith et al., 1983; Kieny et al., 1984; Wiktor et al., 1984).

#### **1.4 Toxoid vaccines**

134 Point Mutation

They are prepared from attenuated strains that are almost or completely devoid of pathogenicity but are capable of inducing a protective immune response. They multiply in the human host and provide continuous antigenic stimulation over a period of time. Primary vaccine failures are uncommon and are usually the result of inadequate storage or administration. Another possibility is interference by related viruses as is suspected in the case of oral polio vaccine in developing countries (Giammanco et al., 1988; Drozdov & Shirman, 1961; Katz & Plotkin, 1968). Several methods have been used to attenuate viruses for vaccine production such as the use of a related virus from another animal (cowpox to prevent smallpox), the administration of pathogenic or partially attenuated virus by an unnatural route, passage of the virus in an "unnatural host" or host cell (e.g. the 17D strain of yellow fever was developed by passage in mice and then in chick embryos (Norrby, 2007) and Polioviruses were passaged in monkey kidney cells (Chezzi et al., 1998) and measles in chick embryo fibroblasts (Katz, 1958), and the development of temperature sensitive

The term killed generally refers to bacterial vaccines, whereas inactivated relates to viral vaccines (Levine et al., 1997). They were the easiest preparations to use. The preparation was simply inactivated. For viruses, the outer virion coat should be left intact but the replicative function should be destroyed. To be effective, non-replicating virus vaccines must contain much more antigen than live vaccines that are able to replicate in the host. Preparation of killed vaccines may take the route of heat or chemicals (Turner et al., 1970). The chemicals used include formaldehyde or beta-propiolactone (Lo Grippo, 1960; Gard, 1960). The traditional agent for inactivation of the virus is formalin (Weil & Gall, 1940; Kim & Sharp, 1967). Excessive treatment with this detergent can destroy immunogenicity whereas insufficient treatment can leave infectious virus capable of causing disease. Soon after the introduction of inactivated polio vaccine, there was an outbreak of paralytic poliomyelitis in the USA due to the distribution of inadequately inactivated polio vaccine (Prevots et al., 1996). This incident led to a review of the formalin inactivation procedure and other inactivating agents are available, such as beta-propiolactone. Another problem was that SV40 was occasionally found as a contaminant and there were fears of the potential

Originally, non-replicating vaccines were derived from crude preparations of virus from animal tissues. As the technology for growing viruses to high titres in cell cultures advanced, it became practical to purify virus and viral antigens. It is now possible to identify the peptide sites encompassing the major antigenic sites of viral antigens, from which highly purified subunit vaccines can be produced. Increasing purification may lead to loss of immunogenicity, and this may necessitate coupling to an immunogenic carrier protein or adjuvant, such as an aluminum salt. Examples of purified subunit vaccines include the HA vaccines for influenza A and B (Bachmayer et al., 1976), and HBsAg derived from the plasma of carriers (Vyas et al., 1984). Subunit vaccines can be further subdivided into those where the antigen is produced using recombinant DNA technology and those

**1.1 Live, attenuated virus vaccines**

mutants (Pringle, 1996).

**1.3 Subunit vaccines**

**1.2 Killed/Inactivated vaccines**

oncogenic nature of the virus (Tam et al., 2004).

based on normal bacteriological growth processes.

Certain pathogens cause disease by secreting an exotoxin: these include tetanus, diphtheria, botulism and cholera. For these bacteria that secrete toxins, or harmful chemicals, a toxoid vaccine might be the answer. These vaccines are used when a bacterial toxin is the main cause of illness. Scientists have found that they can inactivate toxins by treating them with formalin, a solution of formaldehyde and sterilized water. Such "detoxified" toxins, called toxoids, are safe for use in vaccines. When the immune system receives a vaccine containing a harmless toxoid, it learns how to fight off the natural toxin. The immune system produces antibodies that lock onto and block the toxin. Vaccines against diphtheria and tetanus are examples of toxoid vaccines (Bizzini et al., 1970; Alouf, 1987). These vaccines are: safe because they cannot cause the disease they prevent as there is no possibility of reversion to virulence; the vaccine antigens are not actively multiplying, they cannot spread to unimmunized individuals; they are usually stable and long lasting as they are less susceptible to changes in temperature, humidity and light which can result when vaccines are used out in the community.

#### **1.5 Synthetic peptides**

The development of synthetic peptides that might be useful as vaccines depends on the identification of immunogenic sites (Milich, 1990; Hans et al., 2006; Dorothea, 1993; Jonathan, 1987). Synthetic peptide vaccines have been successfully developed for the immunoprophylaxis of infection with foot-and-mouth disease virus (Bittle et al., 1982; Brown, 1990), type A influenza virus (Muller et al., 1982), and poliovirus (Emini et al., 1983). Synthetic peptide vaccines would have many advantages. Their antigens are precisely defined and free from unnecessary components which may be associated with side effects. They are stable and relatively cheap to manufacture. Changes due to natural variation of the virus can be readily accommodated, which would be a great advantage for unstable viruses.

#### **1.6 DNA vaccines**

The demonstration by Wolff and colleagues in 1990 (Wolff et al., 1990) that protein could be expressed following direct inoculation of plasmid DNA into muscle tissue unveiled an

Recombination and Point Mutations in

(Matthijnssens et al., 2008a, 2008b).

Cohen, 1989; Bellamy & Both, 1990).

of this variability upon vaccine development.

Linhares et al., 1999).

Type G Rotavirus Strains: The Challenges of Vaccine Development 137

The high disease burden motivated major efforts to develop a suitable rotavirus vaccine. However, the vaccine efficacy is being challenged by the extensive strain diversity of the rotaviruses (Estes, 2001; Green et al., 1987, 1988; Hoshino et al., 1994; Kapikian et al., 2001;

Reverse-transcription polymerase chain reaction (RT-PCR) is the most widely used method for rotavirus characterization in surveillance studies.  Because of the natural variation in the rotaviral gene sequences, G-type-specificprimer based RT-PCR led to the genotyping failure (Adah et al., 1997; Iturriza- Gómara et al., 2000, 2004a; Maunula & von Bonsdorff, 1998; Rahman et al., 2005b). In addition, the accumulation of point mutations through genetic drift at the type-specic primer binding sites has resulted in failures to type strains or in mistyping. For example, the accumulation of point mutations at the G9 type-specic primer binding site was reported as having an impact for the efficient genotyping of rotaviruses (Martella et al., 2004; Santos et al., 2003). Moreover, some nucleotide identity between genotypes lead to primer cross-reactivity between rotavirus strains as the case of rotavirus G3 and G10 strains using primers developed by Gouvea et al. (1990) as discussed previously (Iturriza-Gómara et al., 2004b).

In order to overcome this problem, a modified classification system for VP4, VP7, and NSP4, and a novel classification system for VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were proposed to be used for international standardization and implementation

Actually, VP4 and VP7 are the main targets for vaccine development strategies. In the present review we will focus on the diversity of VP7 among rotavirus strains and the effect

Similar to most of the group A rotaviruses, the VP7 nucleotide sequence is 1062 nucleotides long. The ORF starts at nucleotide 49 with an AUG (ATG) start codon and ends at nucleotide 1029 with a UAG (TAG) termination codon, comprising 981 nucleotides (Estes &

The gene segment coding for the VP7 glycoprotein is the basis for genotyping group A rotaviruses into at least fifteen G-genotypes. Studies of intragenotype diversity led to subdivision of the G genotypes into several lineages (two major lineages, designated I and II) and sublineages, distinctly identied by unique genomic as well as epidemiological

Genotypes G1, G2, G3, G4 and G9 are the most common G-types in humans (Gentsch et al., 1996; Liprandi et al., 2003; Martella et al., 2003; Okada et al., 2000; Rao et al., 2000; Sereno & Gorziglia, 1994). Nevertheless, over past decades, type G1 rotaviruses have been the most widespread genotype causing acute gastroenteritis in children from many countries covering all continents of the world (Santos & Hoshino, 2005). Type G2 rotavirus represents a different genogroup which appears to have a cyclic pattern of occurrence and yet little information is available about its genetic variability. Type G3 rotavirus have been found in a broad range of host species, including humans, monkeys, dogs, cats, horses, rabbits, mice, sheep and pigs (Martella et al., 2001; Andrej et al., 2008; Hoshino et al., 1984; Paul et al.,

features. Lineage I was further subdivided into four sublineages, Ia–Id.

exciting, new era in vaccinology and gene therapy. DNA-based vaccination offers a number of advantages over other methods of immunization. It is particularly attractive compared to conventional administration of a preformed protein antigen (Ag) because the immunogen is actively synthesized de novo in cells transfected with DNA. The principle of DNA vaccination has been demonstrated for a variety of bacterial, viral and parasitic diseases (Ulmer et al., 1993). Immune responses have been generated by DNA vaccination against a very wide variety of viral, bacterial and protozoal pathogens and toxins (Donnelly et al., 1994; King et al., 1998). Immune responses against influenza viruses have been demonstrated in chickens, mice, ferrets and non-human primates. For humans, the major concern about DNA vaccines is whether the plasmid DNA integrates into the genome randomly, potentially leading to insertional mutagenesis. In addition, the formal acceptance of this novel technology as a new modality of human vaccines depends on the successful demonstration of its safety and efficacy in advanced clinical trials. Several trials evaluated the efficacy of a DNA vaccine targeting human immunodeficiency virus type 1 (HIV-1) for therapeutic and prophylactic applications (MacGregor et al. 1998). However, the results of these early clinical trials were disappointing. The DNA vaccines were safe and well tolerated, but they proved to be poorly immunogenic.

Vaccines may be monovalent (also called univalent) or multivalent (also called polyvalent). A monovalent vaccine is designed to immunize against a single antigen or single microorganism. A multivalent or polyvalent vaccine is designed to immunize against two or more strains of the same microorganism, or against two or more microorganisms. In certain cases a monovalent vaccine may be preferable for rapidly developing a strong immune response.

#### **2. Rotavirus infection and common associated-genotypes**

Infectious acute diarrhea is a significant cause of morbidity and mortality of infants in developing and developed countries and constitutes a major public health problem worldwide. It is estimated that in developing countries (in Africa, Asia and Latin America) 744 million to 1 billion cases of diarrhea and 2.4 to 3.3 million deaths occur annually among children less than 5 years of age, corresponding to 6600 to 9000 deaths per day (Linhares & Bresee, 2000). The viruses are the most common aetiology of these diseases, especially in developed countries, where they cause more than 80% of the cases of acute diarrhea. The most common viral causes of gastroenteritis are rotaviruses and calicivirus (norovirus).

Rotavirus, a member of the *Reoviridae* family, is a highly contagious virus that causes severe and acute dehydrating diarrhea in infants and young children as well as other young animals worldwide (Dhama et al., 2009; Kapikian et al., 2001). Mature rotavirus, nonenveloped virions, contains an 11-segmented, double-stranded RNA (dsRNA) genome enclosed in a triple-layered protein capsid (Hoshino & Kapikian, 2000). The segmented nature of the genome allows rotaviruses to reassort *in vitro* and *in vivo* (Greenberg et al., 1981; Kalica et al., 1981; Gombold & Ramig, 1986).

A dual nomenclature has been used to differentiate rotavirus strains based on their serotype specificities, which are carried by the two outer capsid antigens, VP7 and VP4 (Estes & Kapikian, 2007).

exciting, new era in vaccinology and gene therapy. DNA-based vaccination offers a number of advantages over other methods of immunization. It is particularly attractive compared to conventional administration of a preformed protein antigen (Ag) because the immunogen is actively synthesized de novo in cells transfected with DNA. The principle of DNA vaccination has been demonstrated for a variety of bacterial, viral and parasitic diseases (Ulmer et al., 1993). Immune responses have been generated by DNA vaccination against a very wide variety of viral, bacterial and protozoal pathogens and toxins (Donnelly et al., 1994; King et al., 1998). Immune responses against influenza viruses have been demonstrated in chickens, mice, ferrets and non-human primates. For humans, the major concern about DNA vaccines is whether the plasmid DNA integrates into the genome randomly, potentially leading to insertional mutagenesis. In addition, the formal acceptance of this novel technology as a new modality of human vaccines depends on the successful demonstration of its safety and efficacy in advanced clinical trials. Several trials evaluated the efficacy of a DNA vaccine targeting human immunodeficiency virus type 1 (HIV-1) for therapeutic and prophylactic applications (MacGregor et al. 1998). However, the results of these early clinical trials were disappointing. The DNA vaccines were safe and well

Vaccines may be monovalent (also called univalent) or multivalent (also called polyvalent). A monovalent vaccine is designed to immunize against a single antigen or single microorganism. A multivalent or polyvalent vaccine is designed to immunize against two or more strains of the same microorganism, or against two or more microorganisms. In certain cases a monovalent vaccine may be preferable for rapidly developing a strong immune

Infectious acute diarrhea is a significant cause of morbidity and mortality of infants in developing and developed countries and constitutes a major public health problem worldwide. It is estimated that in developing countries (in Africa, Asia and Latin America) 744 million to 1 billion cases of diarrhea and 2.4 to 3.3 million deaths occur annually among children less than 5 years of age, corresponding to 6600 to 9000 deaths per day (Linhares & Bresee, 2000). The viruses are the most common aetiology of these diseases, especially in developed countries, where they cause more than 80% of the cases of acute diarrhea. The most common viral causes of gastroenteritis are rotaviruses and calicivirus (norovirus).

Rotavirus, a member of the *Reoviridae* family, is a highly contagious virus that causes severe and acute dehydrating diarrhea in infants and young children as well as other young animals worldwide (Dhama et al., 2009; Kapikian et al., 2001). Mature rotavirus, nonenveloped virions, contains an 11-segmented, double-stranded RNA (dsRNA) genome enclosed in a triple-layered protein capsid (Hoshino & Kapikian, 2000). The segmented nature of the genome allows rotaviruses to reassort *in vitro* and *in vivo* (Greenberg et al.,

A dual nomenclature has been used to differentiate rotavirus strains based on their serotype specificities, which are carried by the two outer capsid antigens, VP7 and VP4 (Estes &

tolerated, but they proved to be poorly immunogenic.

1981; Kalica et al., 1981; Gombold & Ramig, 1986).

**2. Rotavirus infection and common associated-genotypes**

response.

Kapikian, 2007).

The high disease burden motivated major efforts to develop a suitable rotavirus vaccine. However, the vaccine efficacy is being challenged by the extensive strain diversity of the rotaviruses (Estes, 2001; Green et al., 1987, 1988; Hoshino et al., 1994; Kapikian et al., 2001; Linhares et al., 1999).

Reverse-transcription polymerase chain reaction (RT-PCR) is the most widely used method for rotavirus characterization in surveillance studies.  Because of the natural variation in the rotaviral gene sequences, G-type-specificprimer based RT-PCR led to the genotyping failure (Adah et al., 1997; Iturriza- Gómara et al., 2000, 2004a; Maunula & von Bonsdorff, 1998; Rahman et al., 2005b). In addition, the accumulation of point mutations through genetic drift at the type-specic primer binding sites has resulted in failures to type strains or in mistyping. For example, the accumulation of point mutations at the G9 type-specic primer binding site was reported as having an impact for the efficient genotyping of rotaviruses (Martella et al., 2004; Santos et al., 2003). Moreover, some nucleotide identity between genotypes lead to primer cross-reactivity between rotavirus strains as the case of rotavirus G3 and G10 strains using primers developed by Gouvea et al. (1990) as discussed previously (Iturriza-Gómara et al., 2004b).

In order to overcome this problem, a modified classification system for VP4, VP7, and NSP4, and a novel classification system for VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were proposed to be used for international standardization and implementation (Matthijnssens et al., 2008a, 2008b).

Actually, VP4 and VP7 are the main targets for vaccine development strategies. In the present review we will focus on the diversity of VP7 among rotavirus strains and the effect of this variability upon vaccine development.

Similar to most of the group A rotaviruses, the VP7 nucleotide sequence is 1062 nucleotides long. The ORF starts at nucleotide 49 with an AUG (ATG) start codon and ends at nucleotide 1029 with a UAG (TAG) termination codon, comprising 981 nucleotides (Estes & Cohen, 1989; Bellamy & Both, 1990).

The gene segment coding for the VP7 glycoprotein is the basis for genotyping group A rotaviruses into at least fifteen G-genotypes. Studies of intragenotype diversity led to subdivision of the G genotypes into several lineages (two major lineages, designated I and II) and sublineages, distinctly identied by unique genomic as well as epidemiological features. Lineage I was further subdivided into four sublineages, Ia–Id.

Genotypes G1, G2, G3, G4 and G9 are the most common G-types in humans (Gentsch et al., 1996; Liprandi et al., 2003; Martella et al., 2003; Okada et al., 2000; Rao et al., 2000; Sereno & Gorziglia, 1994). Nevertheless, over past decades, type G1 rotaviruses have been the most widespread genotype causing acute gastroenteritis in children from many countries covering all continents of the world (Santos & Hoshino, 2005). Type G2 rotavirus represents a different genogroup which appears to have a cyclic pattern of occurrence and yet little information is available about its genetic variability. Type G3 rotavirus have been found in a broad range of host species, including humans, monkeys, dogs, cats, horses, rabbits, mice, sheep and pigs (Martella et al., 2001; Andrej et al., 2008; Hoshino et al., 1984; Paul et al.,

Recombination and Point Mutations in

in several cases of vaccinated infants (CDCP, 1999a).

in many countries such as Finland (Vesikari et al., 2010).

placebo recipients (Vesikari et al., 2006).

immune responses.

Type G Rotavirus Strains: The Challenges of Vaccine Development 139

together with the other 10 genes from an animal rotavirus strain (Midthun & Kapikian, 1996). The next generation of vaccines was formulated to include more than one rotavirus G serotype to provide heterotypic as well as homotypic immunity. The ability of rotaviruses to reassort during mixed infections in vitro allowed the production of reassortant vaccines, termed the "modied Jennerian" approach (Kapikian et al., 1996b). Reassortant viruses contain some genes from the animal rotavirus parent and some genes from the human rotavirus parent. VP7 was thought to be important for protection; therefore, human-animal reassortant rotaviruses for use as vaccines included human VP7 genes to provide protective

*Quadrivalent RRV-based rhesus-human reassortant vaccine.* A RotaShield was the rst multivalent live oral reassortant vaccine (tetravalent, reassortant rhesus-human rotavirus vaccine, [RRV-TV]) contained a mixture of four virus strains representing the most commonly seen G types, G1 to G4: three rhesus-human reassortant strains containing the VP7 genes of human serotypes G1, G2, and G4 strains were substituted for the VP7 gene of the parent RRV, and the fourth strain comprised serotype G3 of rhesus RRV (Kapikian et al., 1996a). RRV-TV was extensively evaluated in eld trials in the United States, Finland, and Venezuela and proved highly effective (80 to 100%) in preventing severe diarrhea due to rotavirus in each of these settings (Joensuu et al., 1997; Perez-Schael et al., 1997; Rennels et al., 1996; Santosham et al., 1997). Due to the proven efficacy, the RRV-TV vaccine was licensed in August 1998 for routine use in children in the United States at 2, 4, and 6 months of age (Centers for Disease Control and Prevention [CDCP], 1999b). Later, this vaccine was withdrawn from the market in 1999 as a consequence of vaccine-associated intussusception

*Pentavalent WC3-based bovine-human reassortant vaccine*. Rotateq®, manufactured by Merck, Inc, USA, is a pentavalent vaccine containing five reassortants representing the common human VP7 types, G1-4 and the most common VP4 type, P[8] (CDCP, 2006). A large efficacy trial with Rotateq® has been completed, which found 74 and 98% efficacy against all and severe disease, respectively and has efficacy against each of the common circulating serotypes. Compared with the rhesus reassortants, the bovine-human reassortants appear to cause less fever while maintaining immunogenicity (Clark et al., 2004). A large safety trial found no evidence of an increased risk of intussusceptions among vacinees compared with

Both RotaTeq and RotaRix have been shown to be effective against rotavirus gastroenteritis, however on March 22, 2010 the Food and Drug Administration (FDA) recommended that the use of the Rotarix vaccine be suspended in the United States because of some DNA from a porcine (pig) virus (porcine circovirus type 1) detected in the vaccine. Subsequently, some DNA from this and another porcine virus were also detected in Rotateq. On May 14, 2010 the FDA updated their recommendations for the use of rotavirus vaccines based on a review of the literature and the input from experts. The RotaTeq vaccine was proven to be effective

Whatever the type of vaccine and strategy of its development, the introduction of a new vaccine faces many hurdles, including cost, production capabilities, safety, and other programmatic issues. For rotavirus vaccines, while there is clearly a need, there are also

additional challenges raised by the emergence of new rotavirus genotypes.

1988; Fitzgerald et al., 1995). The G9 rotavirus was first reported in the United States in the early 1980s (Clark et al., 1987). It represents the fifth most common G genotype of rotavirus infections throughout the world (Gentsch et al., 2005; Santos & Hoshino, 2005, Khamrin et al., 2006).

#### **3. Rotavirus vaccines**

#### **3.1 Monovalent animal and human rotavirus vaccines**