Toll-like receptor 7/8 (TLR7/8) and TLR9 agonists cooperate to enhance HIV-1 envelope antibody responses in rhesus macaques.

UNLABELLED: The development of a vaccine that can induce high titers of functional antibodies against HIV-1 remains a high priority. We have developed an adjuvant based on an oil-in-water emulsion that incorporates Toll-like receptor (TLR) ligands to test whether triggering multiple pathogen-associated molecular pattern receptors could enhance immunogenicity. Compared to single TLR agonists or other pairwise combinations, TLR7/8 and TLR9 agonists combined were able to elicit the highest titers of binding, neutralizing, and antibody-dependent cellular cytotoxicity-mediating antibodies against the protein immunogen, transmitted/founder HIV-1 envelope gp140 (B.63521). We further found that the combination of TLR7/8 and TLR9 agonists was associated with the release of CXCL10 (IP-10), suggesting that this adjuvant formulation may have optimally stimulated innate and adaptive immunity to elicit high titers of antibodies. IMPORTANCE: Combining TLR agonists in an adjuvant formulation resulted in higher antibody levels compared to an adjuvant without TLR agonists. Adjuvants that combine TLR agonists may be useful for enhancing antibody responses to HIV-1 vaccines.

HIV-1 gp120 vaccine induces affinity maturation in both new and persistent antibody clonal lineages.

Most antibodies that broadly neutralize HIV-1 are highly somatically mutated in antibody clonal lineages that persist over time. Here, we describe the analysis of human antibodies induced during an HIV-1 vaccine trial (GSK PRO HIV-002) that used the clade B envelope (Env) gp120 of clone W6.1D (gp120(W6.1D)). Using dual-color antigen-specific sorting, we isolated Env-specific human monoclonal antibodies (MAbs) and studied the clonal persistence of antibodies in the setting of HIV-1 Env vaccination. We found evidence of V(H) somatic mutation induced by the vaccine but only to a modest level (3.8% ± 0.5%; range 0 to 8.2%). Analysis of 34 HIV-1-reactive MAbs recovered over four immunizations revealed evidence of both sequential recruitment of naïve B cells and restimulation of previously recruited memory B cells. These recombinant antibodies recapitulated the anti-HIV-1 activity of participant serum including pseudovirus neutralization and antibody-dependent cell-mediated cytotoxicity (ADCC). One antibody (3491) demonstrated a change in specificity following somatic mutation with binding of the inferred unmutated ancestor to a linear C2 peptide while the mutated antibody reacted only with a conformational epitope in gp120 Env. Thus, gp120(W6.1D) was strongly immunogenic but over four immunizations induced levels of affinity maturation below that of broadly neutralizing MAbs. Improved vaccination strategies will be needed to drive persistent stimulation of antibody clonal lineages to induce affinity maturation that results in highly mutated HIV-1 Env-reactive antibodies.

Impact of immune escape mutations on HIV-1 fitness in the context of the cognate transmitted/founder genome.

BACKGROUND: A modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method. RESULTS: The T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region. CONCLUSIONS: These findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.

Primary infection by a human immunodeficiency virus with atypical coreceptor tropism.

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination.

BACKGROUND: During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection. METHODS AND FINDINGS: To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject. CONCLUSION: The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.

Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce beta-chemokines.

Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to approximately 10 microg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1alpha and MIP-1beta. The release of these beta-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes.