Background: HIV envelope (env) proteins are highly antigenic making them useful for detecting the immune response to natural infection. Envelope protein epitopes are not just virus derived but can be acquired from the host during budding. Some epitopes of host protein beta 2 microglobulin (?2M) become exposed only during incorporation by the virus. These epitopes are defined as cryptic (newly exposed) and are being investigated here, alongside virus-derived envelope protein epitopes for the ability to detect HIV-induced immune responses in vitro. Data obtained in this manner provides information on the potential use of these epitopes as vaccine components or antigens in prognostic/diagnostic assays.
Methods: Synthetic peptides based on epitopes of ?2M (designated as R7V, F7E, S7K, ?2Mp) or env (designated as 2F5, DC1, DV3, and MPER) were synthesized and used in an indirect ELISA to detect antibodies in the serum of HIV infected patients who were treatment-naïve or receiving highly active antiretroviral treatment (HAART). The effect of the peptides on peripheral blood momonuclear cells (PBMCs) was assessed through measuring cytokine secretion (using Cytometric beads) and proliferation (tetrazolium dye uptake, real time cell electronic sensing (RTCES) and flow cytometry using carboxyfluorescein diacetate succimidyl ester (CFSE)). New Zealand white rabbits were immunized with the peptides and affinity purified antibodies prepared and tested for the ability to neutralize viral constructs (pseudo-virus designed to undergo one replication cycle).
Results and Discussion: In individuals infected with HIV-1 subtype C the seroprevalence of antibodies against epitopes of ?2m was variable (between and within experimental groups). ?2M antibodies were prominent (up to 2 fold) in newly infected individuals who were not on treatment or had recently started HAART. One ?2M epitope (R7V) sparked interest in the literature when it repeatedly served as an indicator of non-progression to disease.
In the current study the strongest response was observed for antibodies against V3 loop peptides while antibodies against gp41 epitopes were generally low. At times a notable response was seen against a peptide based on the membrane proximal external region (MPER) of gp41. Proliferation data confirmed that the peptide concentrations used were non-toxic to cells. However, PBMC proliferation was minimal (stimulation index ±1) on all accounts. Tetrazolium dyes are notoriously weak detectors of peptide-induced proliferation, which is why RTCES and flow cytometry were employed as methods with improved levels of sensitivity. Flow cytometry and a fluorescent dye, CFSE, were better detectors of proliferation, allowing the calculation of stimulation indices between 4 and 7. DV3, B2Mp and R7V stimulated proliferation of infected cells. IL-6 (P<0.05) was significantly secreted following stimulation of PBMCs with env antigens. The peptides also stimulated TNF?, IL-17 and IL-10 production, but to a lesser extent. Although the study was designed for the assessment of peptides for characterizing natural infection, the ability of these antigens to elicit an immune response in New Zealand white rabbits, was also evaluated. Titers of >1:6400 and up to 1:152000 were observed. These Polyclonal antibodies (raised against DV3 and MPER) neutralized HIV-1 pseudo-virus ZM53 at >50%.
Conclusion: Antibodies against host-derived beta-2 microglobulin epitopes were reportedly ideal prognostic indicators (prominent in long term non-progressors (LTNP) according to Galea et al, 1996) in infection with HIV-1 subtypes A and B. Margolick et al. (2010) qualified these findings by retrospectively demonstrating that the presence of ?2M epitope antibodies in early infection indicated a tendency toward LTNP. In the current study, using sera collected from HIV-1 subtype C infected individuals; peptides based on ?2M were able to detect antibodies in recently infected individuals and also stimulated cytokine production in vitro. When compared to the viral-derived antigens, the responses detected using host-derived peptides were lower. Although immunogenic, host-derived epitopes do not appear to have value in diagnostic use. The responses to host-derived peptides distinguished recent from later infection, suggesting prognostic potential. The antigenicity of the viral peptides confirmed, what is well reported in the literature, potential use in vaccine development, while the peptides based on host derived protein epitopes showed less ability in this regard.