BackgroundThe RV144 clinical trial showed for the first time that vaccination could provide modest but significant protection from HIV-1 infection. To understand the protective response, and to improve upon the vaccine's efficacy, it is important to define the structure of the immunogens used in the prime/boost regimen. Here we examined the heterogeneity in net charge, attributable to glycoform variation, of the gp120 immunogens contained in the AIDSVAX B/E vaccine.Methodology/Principal FindingsIsoelectric focusing and glycosidase digestion were used to assess variation in net charge of the gp120s contained in the AIDSVAX B/E vaccine used in the RV144 trial. We observed 16 variants of MN-rgp120 and 24 variants of A244-rgp120. Glycoform variation in gp120 produced in Chinese hamster ovary cells was compared to glycoform variation in gp120 produced in the 293F human embryonic kidney cell line, often used for neutralization assays. We found that gp120 variants produced in CHO cells were distinctly more acidic than gp120 variants produced in 293 cells. The effect of glycoform heterogeneity on antigenicity was assessed using monoclonal antibodies. The broadly neutralizing PG9 MAb bound to A244-rgp120, but not to MN-rgp120, whether produced in CHO or in 293. However, PG9 was able to bind with high affinity to MN-rgp120 and A244-rgp120 produced in 293 cells deficient in N-acetylglucosaminyltransferase I.Conclusions/SignificanceMN- and A244-rgp120 used in the RV144 trial exhibited extensive heterogeneity in net charge due to variation in sialic acid-containing glycoforms. These differences were cell line-dependent, affected the antigenicity of recombinant envelope proteins, and may affect assays used to measure neutralization. These studies, together with recent reports documenting broadly neutralizing antibodies directed against carbohydrate epitopes of gp120, suggest that glycoform variation is a key variable to be considered in the production and evaluation of subunit vaccines designed to prevent HIV infection.

RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3′ UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as anti-viral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The anti-viral potential of this conserved element represents a promising start for further research into targeting conserved elements in RNA viruses.

Understanding the determinants of neutralization sensitivity and resistance is important for the development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. In these studies, we have made use of the swarm of closely related envelope protein variants (quasispecies) from an extremely neutralizationresistant clinical isolate in order to identify mutations that conferred neutralization sensitivity to antibodies in sera from HIV-1-infected individuals. Here, we describe a virus with a rare mutation at position 179 in the V2 domain of gp120, where replacement of aspartic acid (D) by asparagine (N) converts a virus that is highly resistant to neutralization by multiple polyclonal and monoclonal antibodies, as well as antiviral entry inhibitors, to one that is sensitive to neutralization. Although the V2 domain sequence is highly variable, D at position 179 is highly conserved in HIV-1 and simian immunodeficiency virus (SIV) and is located within the LDI/V recognition motif of the recently described ␣4␤7 receptor binding site. Our results suggest that the D179N mutation induces a conformational change that exposes epitopes in both the gp120 and the gp41 portions of the envelope protein, such as the CD4 binding site and the MPER, that are normally concealed by conformational masking. Our results suggest that D179 plays a central role in maintaining the conformation and infectivity of HIV-1 as well as mediating binding to ␣4␤7.A major goal in human immunodeficiency virus type 1 (HIV-1) vaccine research is the identification of immunogens able to elicit protective immunity from HIV-1 infection. Results from the recent RV144 clinical trial in Thailand (53) have provided evidence that immunization with vaccines containing the recombinant HIV-1 envelope glycoprotein gp120 (6, 7) can protect humans from HIV infection when incorporated in a prime/boost immunization regimen. Although the level of protection observed in the RV144 trial (31%) was modest, it represents a significant advance in HIV-1 vaccine research and has rekindled the efforts to identify improved subunit vaccine antigens that might achieve even higher levels of protection. In these studies, we have sought to understand the molecular determinants of neutralization sensitivity and resistance in HIV-1 envelope proteins for the purpose of developing improved vaccine antigens.In previous studies (47), we have described a novel method of mutational analysis of the HIV-1 envelope protein, termed swarm analysis, for identification of mutations that confer sensitivity and/or resistance to broadly neutralizing antibodies (bNAbs). This method makes use of the natural amino acid sequence virus variation that occurs in each HIV-infected individual to establish panels of closely related envelope proteins that differ from each other by a limited number of amino acid substitutions. We have previously used this method to identify a novel amino acid substitution in gp41 that conferred sensitivity to neutralization by monoclonal and polyclonal antibodies as we...

Background Recombinant gp120 (MN-rgp120) was a major component of the AIDSVAX B/E vaccine used in the RV144 trial. This was the first clinical trial to show that vaccination could prevent HIV infection in humans. A recent RV144 correlates of protection study found that protection correlated with the presence of antibodies to the V2 domain. It has been proposed that antibodies to the α4β7 binding site in the V2 domain might prevent HIV-1 infection by blocking the ability of virions to recognize α4β7 on activated T-cells. In this study we investigated the specificity of monoclonal antibodies (MAbs) to the V2 domain of MN-rgp120 and examined the possibility that these antibodies could inhibit the binding of MN-rgp120 to the α4β7 integrin. Methodology/Principal Findings Nine MAbs to the V2 domain were isolated from mice immunized with recombinant envelope proteins. The ability of these MAbs to inhibit HIV infection, block the binding of gp120 to CD4, and block the binding of MN-rgp120 to the α4β7 integrin was measured. Mutational analysis showed that eight of the MAbs recognized two immunodominant clusters of amino acids (166–168 and 178–183) located at either end of the C strand within the four-strand anti-parallel sheet structure comprising the V1/V2 domain. Conclusions/Significance These studies showed that the antigenic structure of the V2 domain is exceedingly complex and that MAbs isolated from mice immunized with MN-rgp120 exhibited a high level of strain specificity compared to MAbs to the V2 domain isolated from HIV-infected humans. We found that immunization with MN-rgp120 readily elicits antibodies to the V2 domain and some of these were able to block the binding of MN-rgp120 to the α4β7 integrin.

Serological testing to evaluate antigen-specific antibodies in plasma is generally performed by rapid lateral flow test strips that lack quantitative results or by high complexity immunoassays that are time- and labor-intensive but provide semi-quantitative results. Here, we describe a novel application of biolayer interferometry for the rapid detection of antigen-specific antibody levels in plasma samples, and demonstrate its utility for quantification of SARS-CoV-2 antibodies. Our biolayer interferometry immunosorbent assay (BLI-ISA) utilizes single-use biosensors in an automated “dip-and-read” format, providing real-time optical measurements of antigen loading, plasma antibody binding, and antibody isotype detection. Complete semi-quantitative results are obtained in less than 20 min. BLI-ISA meets or exceeds the performance of high complexity methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and Chemiluminescent Immunoassay. Importantly, our method can be immediately implemented on existing BLI platforms for urgent COVID-19 studies, such as serosurveillance and the evaluation of vaccine candidates. In a broader sense, BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens.