Methods Sixty HIV-uninfected volunteers, randomized into groups of 20 received placebo or 1 mg HIV-DNA intradermally (id) or 3.8 mg intramuscularly (im). DNA plasmids containing HIV-1 gp160 subtypes A, B, C; rev B; p17/p24 gag A, B and RTmut B were given at months 0, 1 and 3 using a needle-free Biojector device. HIV-MVA expressing CRF01_AE HIV-1 env, gag, pol was administered im by needle at months 9 and 21. Sera were tested at baseline, two months post-first and four weeks post-second HIV-MVA boosting. HIV Ab responses were tested using pseudoviruses and TZM-bl cells as well as luciferaseexpressing infectious molecular clones (IMC-LucR) in PBMC-based assays. ADCC responses were tested using the flow cytometry GranToxiLux-based assay.

Background Cellular entry of HIV-1 occurs through fusion at the plasma membrane (PM) or via endocytosis. PM fusion, resulting in deposit of nucleocapsid into the cytoplasm, typically establishes productive infection. In contrast, entry through endocytosis results in either lysosomal degredation, or productive infection through endosomal membrane fusion. While viral entry and permissivity are known to differ between host cells, the influence of these differences on antibody-mediate neutralization is unknown. Here we explore the relationships between HIV-1 entry, neutralization sensitivity, and the mechanisms by which antibodies act.

Background Development of vaccine formulations that induce cross clade neutralizing antibodies to the membrane proximal external region (MPER) of gp41 similar to 4E10 and 2F5 have been extensively investigated. We previously demonstrated that immunization with liposomes containing MPER and lipid A as an adjuvant could induce antibodies to the MPER and IgM monoclonal antibodies obtained from the mice bound to both lipids and MPER and neutralized HIV-1 (Matyas et al., 2009 AIDS 23, 2069-77). We now report a new IgG monoclonal antibody WR324 obtained from mice immunized with a similar vaccine formulation that binds to MPER and can neutralize multiple clades of HIV-1. Methods Mice were immunized with liposomes containing phosphatidyl inositol-4-phosphate and MPER with lipid A as an adjuvant. The peptide, LELDKWASLWNWFDITNWLWYIK (aa 661-684) was derived from gp41 (HXB2 stain). Spleen cells were fused with SP2/O cells and cloned. WR324 was purified and the binding specificities were assessed by ELISA. HIV-1 neutralization was assessed in PBMC using replication competent, Renila reniformis luciferase (LucR)-expressing HIV-1 reporter viruses (LucRIMC) and in a human monocyte derived macrophage (MDM) assay with purified viruses. Results WR324 is an IgG2b with a kappa light chain and it bound to the MPER peptide, but not to lipids or recombinant gp41. WR324 neutralized clade B, BaL and SF162, clade CRF01_AE, CM235, and clade C, GS 014 IMC viruses in the PBMC assay using two different donors. It also neutralized primary viruses, US-1 ,B aL, and clade CRF01_AE, M066 in the MDM assay using 3 different donors. WR324 did not bind to the surface of MDM cells, but binding to HIV-1 was observed by electron microscopy with immuno-gold labeling. Conclusion

Background Antibodies to HIV envelope glycoproteins, with or without neutralization when assayed in standard neutralization assays, are reported to have potential to exert either inhibitory or enhancing effects through interactions with complement and/or Fc receptors. Although most of the standard neutralization assays use free virus rather than carrier-bound HIV as the infectious agent; a cell line as the target for infection; and no complement (C), these conditions may not reflect in vivo events that would include antibody-dependent innate effector mechanisms. Methods We investigated the Fc-receptor-mediated and the complement-mediated antibody-dependent enhancement in trans infection neutralization assays using two broadly neutralizing monoclonal antibodies, 4E10 and b12; primary HIV; PBMC target cells naturally expressing Fc receptors and complement receptors; and with or without complement. Results In the absence of complement, the 4E10 mAb did not neutralize erythrocyte-bound HIV, and the b12 mAb neutralized erythrocyte-bound HIV less effective than the free virus. At low concentrations, 4E10 caused enhancement of infection. In contrast, in the presence of complement, 4E10 neutralized erythrocyte-bound HIV, or caused enhancement of trans infection of erythrocytebound HIV, depending on the mechanism of binding of HIV to erythrocytes. Conclusion Our results are consistent with the concept that the binding of HIV to erythrocytes represents a mechanism of establishing a “safe harbor” for HIV from the adverse effects of antibodies and complement that might otherwise be detrimental to free circulating HIV. Our data shows that broadly neutralizing antibody 4E10 can cause both C-mediated neutralization and enhancement of trans infection. This suggests that erythrocyte-bound HIV-1 serves as a sort of battleground between neutralization and enhancement by antibodies in the presence of C. Because of this, we propose that induction of neutralizing vs. enhancing antibodies can only be differentiated by utilization of a trans infection neutralization assay that might yield more relevant results for in vivo conditions.

To better understand the nature of B cell dysfunctions in subjects infected with HIV-1 subtype A, a rural cohort of 50 treatment-naïve Ugandan patients chronically infected with HIV-1 subtype A was studied, and the relationship between B cell depletion and HIV disease was assessed. B cell absolute counts were found to be significantly lower in HIV-1+ patients, when compared to community matched negative controls (p<0.0001). HIV-1-infected patients displayed variable functional and binding antibody titers that showed no correlation with viral load or CD4+ T cell count. However, B cell absolute counts were found to correlate inversely with neutralizing antibody (NAb) titers against subtype A (p = 0.05) and subtype CRF02_AG (p = 0.02) viruses. A positive correlation was observed between subtype A gp120 binding antibody titers and NAb breadth (p = 0.02) and mean titer against the 10 viruses (p = 0.0002). In addition, HIV-1 subtype A sera showed preferential neutralization of the 5 subtype A or CRF02_AG pseudoviruses, as compared with 5 pseudoviruses from subtypes B, C or D (p<0.001). These data demonstrate that in patients with chronic HIV-1 subtype A infection, significant B cell depletion can be observed, the degree of which does not appear to be associated with a decrease in functional antibodies. These findings also highlight the potential importance of subtype in the specificity of cross-clade neutralization in HIV-1 infection.

Little is known about the neutralization properties of HIV-1 in India to optimally design and test vaccines. For this reason, a functional Env clone was obtained from each of ten newly acquired, heterosexually transmitted HIV-1 infections in Pune, Maharashtra. These clones formed a phylogenetically distinct genetic lineage within subtype C. As Env-pseudotyped viruses the clones were mostly resistant to IgG1b12, 2G12 and 2F5 but all were sensitive to 4E10. When compared to a large multi-subtype panel of Env-pseudotyped viruses (subtypes B, C and CRF02_AG) in neutralization assays with a multi-subtype panel of HIV-1-positive plasma samples, the Indian Envs were remarkably complex. With the exception of the Indian Envs, results of a hierarchical clustering analysis showed a strong subtype association with the patterns of neutralization susceptibility. From these patterns we were able to identify 19 neutralization cluster-associated amino acid signatures in gp120 and 14 signatures in the ectodomain and cytoplasmic tail of gp41. We conclude that newly transmitted Indian Envs are antigenically complex in spite of close genetic similarity. Delineation of neutralization-associated amino acid signatures provides a deeper understanding of the antigenic structure of HIV-1 Env.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.

Open Access Poster presentation P04-28. Breadth and magnitude of neutralizing antibody responses in subjects infected with HIV-1 subtype D or recombinants in Rakai district, Uganda L Eller*1, B Ouma2, P Oballah2, MA Eller1, MS de Souza3, F WabwireMangen4, O Laeyendecker5, T Quinn5, N Kiwanuka4, D Serwadda4, N Sewankambo6, M Wawer5, R Gray5, NL Michael1, ML Robb1, D Montefiori7, BK Brown1 and VR Polonis1