A peptide inhibitor of HIV-1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120.

MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K(d)s ranging from 0.4 to 200 microM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 A resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man(9)GlcNAc(2) (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man(9)GlcNAc(2), and only three of the antibody residues that interact with Man(9)GlcNAc(2) also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.

Safety and efficacy of VRC01 broadly neutralising antibodies in adults with acutely treated HIV (RV397): a phase 2, randomised, double-blind, placebo-controlled trial.

BACKGROUND: HIV-1-specific broadly neutralising antibodies such as VRC01 could promote HIV remission by halting viral replication and clearing infected cells. We investigated whether VRC01 could promote sustained viral control off antiretroviral therapy (ART) in adults who initiated ART during acute HIV infection. METHODS: We did a randomised, double-blind, placebo-controlled trial at the Thai Red Cross AIDS Research Centre in Bangkok, Thailand. Eligible participants were aged 20-50 years, had initiated ART during acute infection (ie, Fiebig stages I-III), had been taking ART for more than 24 months, had fewer than 50 HIV-1 RNA copies per mL on three consecutive measurements, had more than 400 CD4 cells per µL, had fewer than ten copies of integrated HIV-1 DNA per 106 peripheral blood mononuclear cells, and were in generally good health. Eligible participants were randomly assigned (3:1) based on computer-generated lists with a blocking factor of 4 to receive VRC01 (40 mg/kg) or placebo (saline) intravenously every 3 weeks for up to 24 weeks during analytic interruption of ART, followed by continued observation off all therapies. Randomisation was stratified by Fiebig stage (I vs II vs III) at HIV diagnosis. Participants were monitored closely and resumed ART if 1000 or more HIV-1 RNA copies were detected per mL of plasma. The primary outcomes were the frequency of serious adverse events and the proportion of participants with fewer than 50 HIV-1 RNA copies per mL 24 weeks after treatment interruption. Efficacy analyses included all participants who received at least one full dose of study product, and safety analyses included all participants exposed to any study product. The trial was registered with ClinicalTrials.gov, number NCT02664415. This trial is completed. FINDINGS: Between Aug 8, 2016, and Jan 9, 2017, 19 men were randomly assigned, 14 to the VRC01 group and five to the placebo group. One participant in the VRC01 group received a partial infusion without undergoing treatment interruption. The other 18 participants all received at least one full study infusion and underwent ART interruption. No serious adverse events were reported in either group. Only one participant in the VRC01 group achieved the primary efficacy endpoint of viral suppression 24 weeks after ART interruption. The other 17 restarted ART because of a confirmed recording of 1000 or more HIV-1 RNA copies per mL before 24 weeks. INTERPRETATION: VRC01 monotherapy in individuals who initiated ART during acute HIV infection was well tolerated but did not significantly increase the number of participants with viral suppression 24 weeks after ART interruption. Further development of VRC01 and other immunotherapies for HIV will probably occur as part of combination regimens that include several treatments directed against unique therapeutic targets. FUNDING: US Department of the Army, US National Institutes of Health, and the Thai Red Cross AIDS Research Centre.

Systemic and topical use of monoclonal antibodies to prevent the sexual transmission of HIV.

: Passive immunization, the transfer of antibodies to a nonimmune individual to provide immunological protection, has been used for over 100 years to prevent and treat human infectious diseases. The introduction of techniques to produce human mAbs has revolutionized the field, and a large number of human mAbs have been licensed for the treatment of cancer, autoimmune and inflammatory diseases. With the recent discovery and production of highly potent broadly neutralizing and other multifunctional antibodies to HIV, mAbs are now being considered for HIV therapy and prophylaxis. In this review, we briefly present recent advances in the anti-HIV mAb field and outline strategies for the selection, engineering and production of human mAbs, including the modification of their structure for optimized stability and function. We also describe results from nonhuman primate studies and phase 1 clinical trials that have tested the safety, tolerability, pharmacokinetics, and efficacy of mAb-based HIV prevention strategies, and discuss the future of parenteral and topical mAb administration for the prevention of HIV transmission.

Anti-HIV Antibody Responses and the HIV Reservoir Size during Antiretroviral Therapy.

BACKGROUND: A major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the "reservoir"). We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART). METHODS: We evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS) assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR); integrated DNA (Alu PCR); unspliced RNA (rtPCR), multiply-spliced RNA (TILDA), residual plasma HIV RNA (single copy PCR), and replication-competent virus (outgrowth assay). We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR). Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables. RESULTS: Among 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years), the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004) and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003). However, these associations were not statistically significant after a stringent Bonferroni-adjustment of P<0.00045. Multivariate models including age and duration of ART did not markedly alter results. CONCLUSIONS: Our findings suggest that anti-HIV antibody responses may reflect the size of the HIV reservoir during chronic treated HIV disease, possibly via antigen recognition in reservoir sites. Larger, prospective studies are needed to validate the utility of antibody levels as a measure of the total body burden of HIV during treatment.

Passive transfer of neutralizing mAb KD-247 reduces plasma viral load in patients chronically infected with HIV-1.

OBJECTIVE: Neutralizing antibodies against HIV-1 such as a humanized mAb KD-247 can mediate effector functions that attack infected cells in vitro. However, the clinical efficacy of neutralizing antibodies in infected individuals remains to be determined. We evaluated the safety, tolerability and pharmacokinetics of KD-247 infusion and its effect on plasma HIV-1 RNA load and CD4 T-cell count. DESIGN AND METHODS: KD-1002 is a phase Ib, double-blind, placebo-controlled, dose-escalation study of KD-247 in asymptomatic HIV-1 seropositive individuals who did not need antiretroviral therapy. Individuals were randomized to 4, 8 or 16 mg/kg KD-247 or placebo, and received three infusions over a 2-week period. RESULTS: Patients were randomized to receive one of the three doses of KD-247 and the treatment was well tolerated. We observed a significant decrease in HIV RNA in the 8 and 16 mg/kg KD-247 cohorts, with two individuals who achieved more than 1 log reduction of HIV RNA. Two patients in the 16 mg/kg cohort had selections and/or mutations in the V3-tip region that suggested evasion of neutralization. Long-term suppression of viral load was observed in one patient despite a significant decrease in plasma concentration of KD-247, suggesting effects of the antibody other than neutralization or loss of fitness of the evading virus. CONCLUSION: The results indicate that KD-247 reduces viral load in patients with chronic HIV-1 infection and further clinical trials are warranted.

Potent antiretroviral therapy initiates normalization of hypergammaglobulinemia and a decline in HIV type 1-specific antibody responses.

Next to a profound T cell immunodeficiency, HIV-1 infection induces activation and dysfunction of B cells, resulting in hypergammaglobulinemia. Whereas T cell immune reconstitution with potent antiretroviral therapy has been extensively documented, limited data are available on B cell immune reconstitution. We studied the effect of potent antiretroviral therapy on antibody titers to the viral proteins gp120 and p24 and on total IgG concentrations. Three retrospectively chosen groups were studied: a successfully treated group, untreated controls, and subjects with virological failure after several months of successful therapy. In the successfully treated group, the median total IgG concentrations normalized, whereas they remained elevated in the untreated group and rebounded after an initial decline in the therapy failure group. The HIV-1-specific antibody titers declined in the successfully treated group and followed the rebound of the HIV RNA levels in the therapy failure group. With potent antiretroviral therapy the hypergammaglobulinemia normalized whereas HIV-1-specific immune responses were weakened. The weakening of antiviral immunity with therapy may be relevant for current attempts to gain immunological control over the virus through structured treatment interruptions or therapeutic vaccinations.

Virus neutralizing antibodies at different stages of the HIV disease: increased levels after azidothymidine treatment.

Specific HIV-1 neutralizing activity was measured in single serum samples obtained from 52 individuals suffering from different stage of HIV disease, as well as in serum samples collected during a four years follow up of other 13 HIV-1 seropositive persons, from whose seven developed AIDS. Three of these persons were treated with azidothymidine. In the former group of single serum specimens, the specific neutralizing antibody positivity rate was 81 per cent in symptomless persons, 92 per cent in patients with ARC and 43 per cent in patients with AIDS. From 13 HIV-1 infected individuals, prospectively investigated from 1986 to 1990, six remained asymptomatic and no significant fluctuation of specific virus neutralizing antibody levels was noted. During this time period, remaining seven patients developed AIDS. In the sera of AIDS patients, specific neutralizing activity was either not detected or its titres were rather low before the appearance of clinical disease. Three AIDS patients were administered azidothymidine. Specific neutralizing antibody titres increased significantly one month after the beginning of azidothymidine administration and persisted at relatively high levels over several months of follow up.

Monoclonal antibody-based candidate therapeutics against HIV type 1.

Treatment of HIV-1 infection has been highly successful with small molecule drugs. However, resistance still develops. In addition, long-term use can lead to toxicity with unpredictable effects on health. Finally, current drugs do not lead to HIV-1 eradication. The presence of the virus leads to chronic inflammation, which can result in increased morbidity and mortality after prolonged periods of infection. Monoclonal antibodies (mAbs) have been highly successful during the past two decades for therapy of many diseases, primarily cancers and immune disorders. They are relatively safe, especially human mAbs that have evolved in humans at high concentrations to fight diseases and long-term use may not lead to toxicities. Several broadly neutralizing mAbs (bnmAbs) against HIV-1 can protect animals but are not effective when used for therapy of an established infection. We have hypothesized that HIV-1 has evolved strategies to effectively escape neutralization by full-size antibodies in natural infections but not by smaller antibody fragments. Therefore, a promising direction of research is to discover and exploit antibody fragments as potential candidate therapeutics against HIV-1. Here we review several bnmAbs and engineered antibody domains (eAds), their in vitro and in vivo antiviral efficacy, mechanisms used by HIV-1 to escape them, and strategies that could be effective to develop more powerful mAb-based HIV-1 therapeutics.

Association of HIV neutralizing antibody with lower viral load after treatment interruption in a prospective trial (A5170).

OBJECTIVE: We investigated the impact of neutralizing antibodies (NAbs) on CD4 T-cell count and viral load in a cohort of HAART recipients who underwent extended structured treatment interruption. DESIGN: Substudy of NAb in the AIDS Clinical Trials Group 5170 trial. METHODS: Early plasma samples from 50 volunteers who discontinued HAART were evaluated in a peripheral blood mononuclear cell-based neutralization assay against a panel of four subtype B primary isolates. RESULTS: We found that high-titer (90% inhibitory dose > 500) NAb against two or more isolates was associated with reduced viral load (P = 0.003 at 12-week posttreatment interruption). This effect faded with time, losing significance (P = 0.161) by study conclusion. Participants possessing the highest NAb levels against individual isolates appeared more likely to have lower viral loads with the association gaining significance against the R5-tropic primary isolate US1 (P = 0.005). There was no association between broader neutralization and CD4 T-cell slope over time. CONCLUSION: The data suggest that high-titer NAb responses at the time of treatment interruption are associated with reduced viral load over time, but not CD4(+) T-cell decline.

Antibody and B-cell responses may control circulating lipopolysaccharide in patients with HIV infection.

OBJECTIVES: To examine the relationship between plasma markers of microbial translocation and antibodies to lipopolysaccharide (LPS) and circulating memory B cells in patients with HIV infection. DESIGN: Cross-sectional study in antiretroviral therapy (ART)-naive (n = 23) and ART-treated (n = 27) HIV patients. METHODS: Antibodies to LPS and immunoglobulins, assayed in stored serum, and matched memory B-cell counts were correlated with levels of LPS and bacterial 16S ribosome DNA (16S rDNA), assayed in stored plasma. RESULTS: In ART-naive patients, plasma LPS levels correlated inversely with serum levels of IgG and IgA antibodies to LPS (P = 0.03 and 0.006, respectively), serum levels of IgA anti-LPS correlated with total IgA (P < 0.0001) and levels of IgG anti-LPS correlated with IgM(+) memory B-cell counts (P = 0.025). In ART-treated patients, plasma LPS levels were not related to levels of LPS antibodies, but were related to CD4(+) T-cell and switched memory B-cell counts. There were no correlations with plasma levels of 16S rDNA. CONCLUSION: Plasma LPS levels were associated with antibody and possibly B-cell responses to LPS in ART-naive HIV patients, whereas they were associated with the degree of immune reconstitution in ART-treated patients.

A novel strategy for inducing enhanced mucosal HIV-1 antibody responses in an anti-inflammatory environment.

Prophylactic vaccination against HIV-1 sexual transmission will probably require antibody elicitation at genital mucosal surfaces. However, HIV-1 envelope glycoprotein (Env)-based antigens are weakly immunogenic, particularly when applied mucosally. The polyanion PRO 2000 is safe for human vaginal application, and thus may represent a potential formulating agent for vaginal delivery of experimental vaccine immunogens. Based upon its biochemical properties, we hypothesized that PRO 2000 might enhance mucosal immunogenicity of HIV-1 envelope glycoprotein (Env)-based antigens, promoting local and systemic immune responses. Vaginal immunization with Env-PRO 2000 resulted in significantly increased titres of Env-specific mucosal IgA and IgG in mice and rabbits, respectively, compared to Env alone, revealing modest but significant mucosal adjuvant activity for PRO 2000. In vitro, PRO 2000 associated with Env, protecting the glycoprotein from proteolytic degradation in human vaginal lavage. Unexpectedly, PRO 2000 antagonized TLR4 activation, suppressing local production of inflammatory cytokines. Since inflammation-mediated recruitment of viral target cells is a major risk factor in HIV-1 transmission, the immune modulatory and anti-inflammatory activities of PRO 2000 combined with its intravaginal safety profile suggests promise as an HIV-1 mucosal vaccine formulating agent.

Increase in neutralizing antibody titer against sequential autologous HIV-1 isolates after 16 weeks saquinavir (Invirase) treatment.

The humoral immune response to HIV infection plays an important role in determining disease progression. Few and discordant results correlate changes in neutralizing antibody (NtAb) titer with antiretroviral treatment. The NtAb titer against autologous-HIV was evaluated in 33 patients treated with the protease inhibitor saquinavir (SQV, Invirase) and zidovudine (ZDV) alone or in combination. Ten out of 33 (30%) patients showed a significant increase (4-fold or greater) in NtAb titer from baseline in response to the initiation of therapy. A significant correlation (P = 0.007) was found between an increase in NtAb titer and treatment with SQV alone (5 subjects) or in combination (5 subjects). A significant decrease in NtAb titer was detected in 7 patients, 5 of whom were treated with ZDV alone. After one year of therapy a significant decrease in HIV-RNA copy number (> 0.5 log) with respect to baseline value was detected only in patients treated with SQV alone or in combination. Patients with increased NtAb titer showed a significantly reduced HIV-RNA copy number and increased CD4+ cell count at week 16 of treatment which were sustained up to week 52. These data suggest that treatment with SQV can improve neutralizing activity against autologous virus as well as bring about a significant and sustained reduction in viral load.