Comparative Immunogenicity of HIV-1 Clade C Envelope Proteins for Prime/Boost Studies

BACKGROUND: Previous clinical efficacy trials failed to support the continued development of recombinant gp120 (rgp120) as a candidate HIV vaccine. However, the recent RV144 HIV vaccine trial in Thailand showed that a prime/boost immunization strategy involving priming with canarypox vCP1521 followed by boosting with rgp120 could provide significant, although modest, protection from HIV infection. Based on these results, there is renewed interest in the development of rgp120 based antigens for follow up vaccine trials, where this immunization approach can be applied to other cohorts at high risk for HIV infection. Of particular interest are cohorts in Africa, India, and China that are infected with clade C viruses. METHODOLOGY/PRINCIPAL FINDINGS: A panel of 10 clade C rgp120 envelope proteins was expressed in 293 cells, purified by immunoaffinity chromatography, and used to immunize guinea pigs. The resulting sera were collected and analyzed in checkerboard experiments for rgp120 binding, V3 peptide binding, and CD4 blocking activity. Virus neutralization studies were carried out with two different assays and two different panels of clade C viruses. A high degree of cross reactivity against clade C and clade B viruses and viral proteins was observed. Most, but not all of the immunogens tested elicited antibodies that neutralized tier 1 clade B viruses, and some sera neutralized multiple clade C viruses. Immunization with rgp120 from the CN97001 strain of HIV appeared to elicit higher cross neutralizing antibody titers than the other antigens tested. CONCLUSIONS/SIGNIFICANCE: While all of the clade C antigens tested were immunogenic, some were more effective than others in eliciting virus neutralizing antibodies. Neutralization titers did not correlate with rgp120 binding, V3 peptide binding, or CD4 blocking activity. CN97001 rgp120 elicited the highest level of neutralizing antibodies, and should be considered for further HIV vaccine development studies

Anti-V3 Monoclonal Antibodies Display Broad Neutralizing Activities against Multiple HIV-1 Subtypes

Background: The V3 loop of the HIV-1 envelope (Env) glycoprotein gp120 was identified as the “principal neutralizing domain” of HIV-1, but has been considered too variable to serve as a neutralizing antibody (Ab) target. Structural and immunochemical data suggest, however, that V3 contains conserved elements which explain its role in binding to virus co-receptors despite its sequence variability. Despite this evidence of V3 conservation, the ability of anti-V3 Abs to neutralize a significant proportion of HIV-1 isolates from different subtypes (clades) has remained controversial. Methods: HIV-1 neutralization experiments were conducted in two independent laboratories to test human anti-V3 monoclonal Abs (mAbs) against pseudoviruses (psVs) expressing Envs of diverse HIV-1 subtypes from subjects with acute and chronic infections. Neutralization was defined by 50% inhibitory concentrations (IC50), and was statistically assessed based on the area under the neutralization titration curves (AUC). Results: Using AUC analyses, statistically significant neutralization was observed by ≥1 anti-V3 mAbs against 56/98 (57%) psVs expressing Envs of diverse subtypes, including subtypes A, AG, B, C and D. Even when the 10 Tier 1 psVs tested were excluded from the analysis, significant neutralization was detected by ≥1 anti-V3 mAbs against 46/88 (52%) psVs from diverse HIV-1 subtypes. Furthermore, 9/24 (37.5%) Tier 2 viruses from the clade B and C standard reference panels were neutralized by ≥1 anti-V3 mAbs. Each anti-V3 mAb tested was able to neutralize 28–42% of the psVs tested. By IC50 criteria, 40/98 (41%) psVs were neutralized by ≥1 anti-V3 mAbs. Conclusions: Using standard and new statistical methods of data analysis, 6/7 anti-V3 human mAbs displayed cross-clade neutralizing activity and revealed that a significant proportion of viruses can be neutralized by anti-V3 Abs. The new statistical method for analysis of neutralization data provides many advantages to previously used analyses

Unusual Cysteine Content in V1 Region of gp120 From an Elite Suppressor That Produces Broadly Neutralizing Antibodies

Although it is now possible to produce recombinant HIV envelope glycoproteins (Envs) with epitopes recognized by the 5–6 major classes of broadly neutralizing antibodies (bNAbs), these have failed to consistently stimulate the formation of bNAbs in immunized animals or humans. In an effort to identify new immunogens better able to elicit bNAbs, we are studying Envs derived from rare individuals who possess bNAbs and are able to control their infection without the need for anti-retroviral drugs (elite supressors or ES), hypothesizing that in at least some people the antibodies may mediate durable virus control. Because virus evolution in people with the ES only phenotype was reported to be limited, we reasoned the Env proteins recovered from these individuals may more closely resemble the Envs that gave rise to bNAbs compared to the highly diverse viruses isolated from normal progressors. Using a phenotypic assay, we screened 25 controllers and identified two for more detailed investigation. In this study, we examined 20 clade B proviral sequences isolated from an African American woman, who had the rare bNAb/ES phenotype. Phylogenetic analysis of proviral envelope sequences demonstrated low genetic diversity. Envelope proteins were unusual in that most possessed two extra cysteines within an elongated V1 region. In this report, we examine the impact of the extra cysteines on the binding to bNAbs, virus infectivity, and sensitivity to neutralization. These data suggest structural motifs in V1 can affect infectivity, and that rare viruses may be prevented from developing escape

Constraints on HIV-1 evolution and immunodominance revealed in monozygotic adult twins infected with the same virus

The predictability of virus–host interactions and disease progression in rapidly evolving human viral infections has been difficult to assess because of host and genetic viral diversity. Here we examined adaptive HIV-specific cellular and humoral immune responses and viral evolution in adult monozygotic twins simultaneously infected with the same virus. CD4 T cell counts and viral loads followed similar trajectories over three years of follow up. The initial CD8 T cell response targeted 17 epitopes, 15 of which were identical in each twin, including two immunodominant responses. By 36 months after infection, 14 of 15 initial responses were still detectable in both, whereas all new responses were subdominant and remained so. Of four responses that declined in both twins, three demonstrated mutations at the same residue. In addition, the evolving antibody responses cross-neutralized the other twin's virus, with similar changes in the pattern of evolution in the envelope gene. These results reveal considerable concordance of adaptive cellular and humoral immune responses and HIV evolution in the same genetic environment, suggesting constraints on mutational pathways to HIV immune escape

Chimeric Rhinoviruses Displaying MPER Epitopes Elicit Anti-HIV Neutralizing Responses

Background: The development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER) of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV. Methodology/Principle Findings: Guided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV) displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPERdisplaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested. Conclusions: Optimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection

Broadly neutralizing antibody responses in a large longitudinal sub-Saharan HIV primary infection cohort

Author Summary Understanding how HIV-1-broadly neutralizing antibodies (bnAbs) develop during natural infection is essential to the design of an efficient HIV vaccine. We studied kinetics and correlates of neutralization breadth in a large sub-Saharan African longitudinal cohort of 439 participants with primary HIV-1 infection. Broadly nAb responses developed in 15% of individuals, on average three years after infection. Broad neutralization was associated with high viral load, low CD4+ T cell counts, virus subtype C infection and HLA*A3(-) genotype. A correlation with high overall plasma IgG levels and anti-Env binding titers was also found. Specificity mapping of the bnAb responses showed that glycan-dependent epitopes, in particular the N332 region, were most commonly targeted, in contrast to other bnAb epitopes, suggesting that the HIV Env N332-glycan epitope region may be a favorable target for vaccine design

Broad Antibody Mediated Cross-Neutralization and Preclinical Immunogenicity of New Codon-Optimized HIV-1 Clade CRF02_AG and G Primary Isolates

Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary “street strain” isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G

Sex Disparities and Neutralizing-Antibody Durability to SARS-CoV-2 Infection in Convalescent Individuals

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has now caused over 2 million deaths worldwide and continues to expand. Currently, much is unknown about functionally neutralizing human antibody responses and durability to SARS-CoV-2 months after infection or the reason for the discrepancy in COVID-19 disease and sex. Using convalescent-phase sera collected from 101 COVID-19-recovered individuals 21 to 212 days after symptom onset with 48 additional longitudinal samples, we measured functionality and durability of serum antibodies. We also evaluated associations of individual demographic and clinical parameters with functional neutralizing antibody responses to COVID-19. We found robust antibody durability out to 6 months, as well as significant positive associations with the magnitude of the neutralizing antibody response and male sex and in individuals with cardiometabolic comorbidities. IMPORTANCE In this study, we found that neutralizing antibody responses in COVID-19-convalescent individuals vary in magnitude but are durable and correlate well with receptor binding domain (RBD) Ig binding antibody levels compared to other SARS-CoV-2 antigen responses. In our cohort, higher neutralizing antibody titers are independently and significantly associated with male sex compared to female sex. We also show for the first time that higher convalescent antibody titers in male donors are associated with increased age and symptom grade. Furthermore, cardiometabolic comorbidities are associated with higher antibody titers independently of sex. Here, we present an indepth evaluation of serologic, demographic, and clinical correlates of functional antibody responses and durability to SARS-CoV-2 which supports the growing literature on sex discrepancies regarding COVID-19 disease morbidity and mortality, as well as functional neutralizing antibody responses to SARS-CoV-2

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report II

BACKGROUND: Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). METHODS: In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. FINDINGS: Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. CONCLUSIONS: Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation