Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein

BACKGROUND: Of the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat. RESULTS: Tat proteins obtained using the novel strategy described here were free of contaminants and retained biological functions as evaluated in a range of assays including the induction of cytokines, upregulation of chemokine coreceptor, transactivation of the viral promoter and rescue of a Tat-defective virus. Given the highly unstable nature of Tat, we evaluated the effect of the storage conditions on the biological function of Tat following purification. Tat stored in a lyophilized form retained complete biological activity regardless of the storage temperature. To understand if variations in the primary structure of Tat could influence the secondary structure of the protein and consequently its biological functions, we determined the CD spectra of subtype-C and -B Tat proteins. We demonstrate that subtype-C Tat may have a relatively higher ordered structure and be less flexible than subtype-B Tat. We show that subtype-C Tat as a protein, but not as a DNA expression vector, was consistently inferior to subtype-B Tat in a variety of biological assays. Furthermore, using ELISA, we evaluated the anti-Tat antibody titers in a large number of primary clinical samples (n = 200) collected from all four southern Indian states. Our analysis of the Indian populations demonstrated that Tat is non-immunodominant and that a large variation exists in the antigen-specific antibody titers. CONCLUSION: Our report not only describes a simple protein purification strategy for Tat but also demonstrates important structural and functional differences between subtype-B and -C Tat proteins. Furthermore, this is the first report of protein purification and characterization of subtype-C Tat

Exposure to Apoptotic Activated CD4+ T Cells Induces Maturation and APOBEC3G- Mediated Inhibition of HIV-1 Infection in Dendritic Cells

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4+ T cells (ApoInf) or apoptotic uninfected activated CD4+ T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4+ T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4+ T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection

Loss of marginal zone B-cells in SHIVSF162P4 challenged rhesus macaques despite control of viremia to low or undetectable levels in chronic infection

AbstractMarginal zone (MZ) B cells generate T-independent antibody responses to pathogens before T-dependent antibodies arise in germinal centers. They have been identified in cynomolgus monkeys and monitored during acute SIV infection, yet have not been well-studied in rhesus macaques. Here we characterized rhesus macaque MZ B cells, present in secondary lymphoid tissue but not peripheral blood, as CD19+, CD20+, CD21hi, IgM+, CD22+, CD38+, BTLA+, CD40+, CCR6+ and BCL-2+. Compared to healthy macaques, SHIVSF162P4-infected animals showed decreased total B cells and MZ B cells and increased MZ B cell Ki-67 expression early in chronic infection. These changes persisted in late chronic infection, despite viremia reductions to low or undetectable levels. Expression levels of additional phenotypic markers and RNA PCR array analyses were in concert with continued low-level activation and diminished function of MZ B cells. We conclude that MZ B-cell dysregulation and dysfunction associated with SIV/HIV infection are not readily reversible

Mucosal B Cells Are Associated with Delayed SIV Acquisition in Vaccinated Female but Not Male Rhesus Macaques Following SIVmac251 Rectal Challenge.

Many viral infections, including HIV, exhibit sex-based pathogenic differences. However, few studies have examined vaccine-related sex differences. We compared immunogenicity and protective efficacy of monomeric SIV gp120 with oligomeric SIV gp140 in a pre-clinical rhesus macaque study and explored a subsequent sex bias in vaccine outcome. Each immunization group (16 females, 8 males) was primed twice mucosally with replication-competent Ad-recombinants encoding SIVsmH4env/rev, SIV239gag and SIV239nefΔ1-13 and boosted twice intramuscularly with SIVmac239 monomeric gp120 or oligomeric gp140 in MF59 adjuvant. Controls (7 females, 5 males) received empty Ad and MF59. Up to 9 weekly intrarectal challenges with low-dose SIVmac251 were administered until macaques became infected. We assessed vaccine-induced binding, neutralizing, and non-neutralizing antibodies, Env-specific memory B cells and plasmablasts/plasma cells (PB/PC) in bone marrow and rectal tissue, mucosal Env-specific antibodies, and Env-specific T-cells. Post-challenge, only one macaque (gp140-immunized) remained uninfected. However, SIV acquisition was significantly delayed in vaccinated females but not males, correlated with Env-specific IgA in rectal secretions, rectal Env-specific memory B cells, and PC in rectal tissue. These results extend previous correlations of mucosal antibodies and memory B cells with protective efficacy. The gp140 regimen was more immunogenic, stimulating elevated gp140 and cyclic V2 binding antibodies, ADCC and ADCP activities, bone marrow Env-specific PB/PC, and rectal gp140-specific IgG. However, immunization with gp120, the form of envelope immunogen used in RV144, the only vaccine trial to show some efficacy, provided more significant acquisition delay. Further over 40 weeks of follow-up, no gp120 immunized macaques met euthanasia criteria in contrast to 7 gp140-immunized and 2 control animals. Although males had higher binding antibodies than females, ADCC and ADCP activities were similar. The complex challenge outcomes may reflect differences in IgG subtypes, Fc glycosylation, Fc-R polymorphisms, and/or the microbiome, key areas for future studies. This first demonstration of a sex-difference in SIV vaccine-induced protection emphasizes the need for sex-balancing in vaccine trials. Our results highlight the importance of mucosal immunity and memory B cells at the SIV exposure site for protection

Comparative immunogenicity of monomeric gp120 and oligomeric gp140: mucosal and bone marrow responses.

<p>Immune responses were evaluated on mucosal secretions and bone marrow samples obtained at week 53, 2 weeks following the second Env protein immunization.</p><p>^ang specific/μg total.</p><p>^bPercent Env-specific ASC relative to total ASC in bone marrow.</p><p>Comparative immunogenicity of monomeric gp120 and oligomeric gp140: mucosal and bone marrow responses.</p

Comparison of immune responses between female and male macaques.

<p>Binding antibody titers to SIV_mac239 gp120 and gp140 over the course of immunization and following infection in (A) gp120- and (B) gp140-immunized female and male macaques. (C) Binding antibody titers by sex of combined gp120- and gp140 immunization groups to SIV_mac239 gp120 over the course of immunization and 2wkpi. (D) Binding antibody titers to SIV_E660 gp120 over the course of immunization in females and males of combined gp120- and gp140-immunization groups. Pre-bleed samples were not tested but binding titers of control macaque samples at all time points were <50. Titers are expressed as geometric means with 95% CL. (E) Serum phagocytic activity (phagocytosis score/background phagocytosis) to gp140 targets 2wkpi in females and males of the gp120- and gp140 immunization groups. (F) Serum ADCC activity of female and male macaques to gp120 and gp140 targets by immunization group at wk 53. (G) Rectal gp120-specific memory B cells (identified by flow cytometry) in female and male macaques of combined gp120- and gp140-immunization groups at 2 wk post-second Env boost (wk 53), 2wkpi, and 8wkpi. Mean values ± SEM are shown in E and G. One gp140-immunized macaque remained uninfected, and is omitted from 2wkpi analyses. In panel G, rectal samples of 15 macaques (6 from each gp120- and gp140-immunization group and 3 controls) are not shown at wk 53 as samples were lost due to a processing error.</p

Immunological correlates of delayed SIV acquisition.

<p>Influence of rectal Env-specific IgA at wk 55 on the rate of infection in (A) all immunized macaques, (B) in gp120 immunized macaques, and (C) in gp140 immunized macaques. Influence of rectal Env-specific IgA at wk 55 on the rate of infection in (D) all immunized females, (E) in gp120-immunized females, and (F) in gp140-immunized females. gp120 and gp140-immunized macaques were tested against gp120 and gp140 proteins respectively. Control background levels were subtracted prior to analysis. Correlation analysis of Env-specific memory B cells in rectal tissue identified by flow cytometry 2wkpi with number of challenges to become infected in (G) all immunized females, (H) all immunized males, (I) gp120-immunized females, and (J) gp140-immunized females. Correlation analysis of rectal plasma cells identified by flow cytometry 2wkpi with number of challenges to become infected in (K) all immunized females, (L) all immunized males, (M) gp120-immunized females, and (N) gp140-immunized females. One gp140-immunized female remained uninfected and is excluded from the 2wkpi time point in panels G, J, K, and N. In panels K-N, PC were identified following the first challenge using IRF-4 and BCL2 intracellular markers. Subsequently, all data were obtained using the surface marker CD138 and IRF-4. As the two approaches are not comparable, the week one challenge data are omitted.</p

Comparative immunogenicity of monomeric gp120 and oligomeric gp140: mucosal and bone marrow responses.

<p>Immune responses were evaluated on mucosal secretions and bone marrow samples obtained at week 53, 2 weeks following the second Env protein immunization.</p><p>^ang specific/μg total.</p><p>^bPercent Env-specific ASC relative to total ASC in bone marrow.</p><p>Comparative immunogenicity of monomeric gp120 and oligomeric gp140: mucosal and bone marrow responses.</p