Amino acid substitution of the membrane-proximal external region alter neutralization sensitivity in a chronic HIV-1 clade B infected patient.

The membrane-proximal external region (MPER) represents a highly conserved region of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (env) targeted by several broadly neutralizing antibodies (bnAbs). In this study, we employed single genome amplification to amplify 34 full-length env sequences from the 2005 plasma sample of CBJC504, a chronic HIV-1 clade B infected individual. We identified three amino acid changes (N671S, D674N, and K677R) in the MPER. A longitudinal analysis revealed that the proportion of env sequences with MPER mutations increased from 26.5 % in 2005 to 56.0 % in 2009, and the sequences with the same mutation clustered together. Nine functional pseudoviruses were generated from the 34 env sequences to examine the effect of these mutations on neutralizing activity. Pseudoviruses carrying N674 or R677 mutations demonstrate increased sensitivity to autologous plasma and monoclonal antibodies 2F5, 4E10, and 10E8. Reverse mutations were performed in env including N674, R677, D659, and S671/N677 mutations, to validate the impact of the mutations on neutralizing sensitivity. Neutralization assays indicated that the N671S mutation increased neutralization sensitivity to 2F5 and 10E8. The amino acid R at position 677 increased viral resistance to 10E8, whereas N enhanced viral resistance to 4E10 and 10E8. It has been proposed that critical amino acids in the extra-MPER and the number of potential N-like glycosylation sites (PNGSs) in the V1 loop may have an impact on neutralizing activity. Understanding the mutations and evolution of MPER in chronically infected patients with HIV-1 is crucial for the design and development of vaccines that trigger bnAbs against MPER.

Neutralization Sensitivity and Evolution of Virus in a Chronic HIV-1 Clade B Infected Patient with Neutralizing Activity against Membrane-Proximal External Region.

The membrane-proximal external region (MPER) is a promising HIV-1 vaccine target owing to its linear neutralizing epitopes and highly conserved amino acids. Here, we explored the neutralization sensitivity and investigated the MPER sequences in a chronic HIV-1 infected patient with neutralizing activity against the MPER. Using single-genome amplification (SGA), 50 full-length HIV-1 envelope glycoprotein (env) genes were isolated from the patient's plasma at two time points (2006 and 2009). The neutralization sensitivity of 14 Env-pseudoviruses to autologous plasma and monoclonal antibodies (mAbs) was evaluated. Env gene sequencing revealed that the diversity of Env increased over time and four mutation positions (659D, 662K, 671S, and 677N/R) were identified in the MPER. The K677R mutation increased the IC50 values of pseudoviruses approximately twofold for 4E10 and 2F5, and E659D increased the IC50 up to ninefold for 4E10 and fourfold for 2F5. These two mutations also decreased the contact between gp41 and mAbs. Almost all mutant pseudoviruses were resistant to autologous plasma at both the earlier and concurrent time points. Mutations 659D and 677R in the MPER decreased the neutralization sensitivity of Env-pseudoviruses, providing a detailed understanding of MPER evolution which might facilitate advances in the design of HIV-1 vaccines.

Functional properties of the spike glycoprotein of the emerging SARS-CoV-2 variant B.1.1.529.

The recently emerged B.1.1.529 (Omicron) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant has a highly divergent spike (S) glycoprotein. We compared the functional properties of B.1.1.529 BA.1 S with those of previous globally prevalent SARS-CoV-2 variants, D614G and B.1.617.2. Relative to these variants, B.1.1.529 S exhibits decreases in processing, syncytium formation, virion incorporation, and ability to mediate infection of cells with high TMPRSS2 expression. B.1.1.529 and B.1.617.2 S glycoproteins bind ACE2 with higher affinity than D614G S. The unliganded B.1.1.529 S trimer is less stable at low temperatures than the other SARS-CoV-2 Ss, a property related to its more "open" S conformation. Upon ACE2 binding, the B.1.1.529 S trimer sheds S1 at 37°C, but not at 0°C. B.1.1.529 pseudoviruses are relatively resistant to neutralization by sera from patients with coronavirus disease 2019 (COVID-19) and vaccinees. These properties of the B.1.1.529 S glycoprotein likely influence the transmission, cytopathic effects, and immune evasion of this emerging variant.

HIV-2 Neutralization Sensitivity in Relation to Co-Receptor Entry Pathways and Env Motifs.

HIV-2, compared to HIV-1, elicits potent and broadly neutralizing antibodies, and uses a broad range of co-receptors. However, both sensitivity to neutralization and breadth of co-receptor use varies between HIV-2 isolates, and the molecular background is still not fully understood. Thus, in the current study, we have deciphered relationships between HIV-2 neutralization sensitivity, co-receptor use and viral envelope glycoprotein (Env) molecular motifs. A panel of primary HIV-2 isolates, with predefined use of co-receptors, was assessed for neutralization sensitivity using a set of HIV-2 Env-directed monoclonal antibodies and co-receptor indicator cell lines. Neutralization sensitivity of the isolates was analysed in relation target cell co-receptor expression, in addition to amino acid motifs and predicted structures of Env regions. Results showed that HIV-2 isolates were more resistant to neutralizing antibodies when entering target cells via the alternative co-receptor GPR15, as compared to CCR5. A similar pattern was noted for isolates using the alternative co-receptor CXCR6. Sensitivity to neutralizing antibodies appeared also to be linked to specific Env motifs in V1/V2 and C3 regions. Our findings suggest that HIV-2 sensitivity to neutralization depends both on which co-receptor is used for cell entry and on specific Env motifs. This study highlights the multifactorial mechanisms behind HIV-2 neutralization sensitivity.

Neutralization Sensitivity of HIV-1 CRF07_BC From an Untreated Patient With a Focus on Evolution Over Time.

The diversity of HIV-1 envelope (Env) glycoproteins affects the potency and breadth of broadly neutralizing antibodies (bNAbs), a promising alternative to antiretroviral drugs for the prevention and treatment of HIV-1 infection. To facilitate immunogen design and development of therapeutic neutralizing antibodies, we characterized viral evolution and monitored the changes in neutralizing activity/sensitivity of a long-term non-progressor patient with HIV-1 CRF07_BC infection. Fifty-nine full-length Env gene fragments were derived from four plasma samples sequentially harvested from the patient between 2016 and 2020. Sequencing of patient-derived Env genes revealed that potential N-linked glycosylation sites (PNGS) in V1 and V5 significantly increased over time. Further, 24 functional Env-pseudotyped viruses were generated based on Env gene sequences. While all 24 Env-pseudotyped viruses remained sensitive to concurrent and subsequent autologous plasma, as well as bNAbs, including 10E8, VRC01, and 12A21, Env-pseudotyped viruses corresponding to later sampling time were increasingly more resistant to autologous plasma and bNAbs. All 24 Env-pseudotyped viruses were resistant to bNAbs 2G12, PGT121, and PGT135. The neutralization breadth of plasma from all four sequential samples was 100% against the global HIV-1 reference panel. Immune escape mutants resulted in increased resistance to bNAb targeting of different epitopes. Our study identified known mutations F277W in gp41 and previously uncharacterized mutation S465T in V5 which may be associated with increased viral resistance to bNAbs.

Concomitant Enhancement of HIV-1 Replication Potential and Neutralization-Resistance in Concert With Three Adaptive Mutations in Env V1/C2/C4 Domains.

HIV-1 Env protein functions in the entry process and is the target of neutralizing antibodies. Its intrinsically high mutation rate is certainly one of driving forces for persistence/survival in hosts. For optimal replication in various environments, HIV-1 Env must continue to adapt and evolve through balancing sometimes incompatible function, replication fitness, and neutralization sensitivity. We have previously reported that adapted viruses emerge in repeated and prolonged cultures of cells originally infected with a macaque-tropic HIV-1NL4-3 derivative. We have also shown that the adapted viral clones exhibit enhanced growth potentials both in macaque PBMCs and individuals, and that three single-amino acid mutations are present in their Env V1/C2/C4 domains. In this study, we investigated how lab-adapted and highly neutralization-sensitive HIV-1NL4-3 adapts its Env to macaque cells with strongly replication-restrictive nature for HIV-1. While a single and two mutations gave a significantly enhanced replication phenotype in a macaque cell line and also in human cell lines that stably express either human CD4 or macaque CD4, the virus simultaneously carrying the three adaptive mutations always grew best. Entry kinetics of parental and triple mutant viruses were similar, whereas the mutant was significantly more readily inhibited for its infectivity by soluble CD4 than parental virus. Furthermore, molecular dynamics simulations of the Env ectodomain (gp120 and gp41 ectodomain) bound with CD4 suggest that the three mutations increase binding affinity of Env for CD4 in solution. Thus, it is quite likely that the affinity for CD4 of the mutant Env is enhanced relative to the parental Env. Neutralization sensitivity of the triple mutant to CD4 binding site antibodies was not significantly different from that of parental virus, whereas the mutant exhibited a considerably higher resistance against neutralization by a CD4-induced epitope antibody and Env trimer-targeting V1/V2 antibodies. These results suggest that the three adaptive mutations cooperatively promote viral growth via increased CD4 affinity, and also that they enhance viral resistance to several neutralization antibodies by changing the Env-trimer conformation. In total, we have verified here an HIV-1 adaptation pathway in host cells and individuals involving Env derived from a lab-adapted and highly neutralization-sensitive clone.

Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors.

BACKGROUND: HIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors. RESULTS: Mutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the "conformational unmasking" of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples. CONCLUSIONS: Mutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy.