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Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies., Competing Interests: Sophia W. Li and Phillip W. Berman have filed a patent on C1s‐deficient cells for the production of vaccines and biopharmaceutical proteins. The patent is entitled “Complement Component 1s (C1s) Deficient Cells for Production of Vaccines and Biopharmaceutical Proteins” with publication Number WO/2020/081328. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A major challenge in HIV vaccine development is the identification of immunogens able to elicit broadly neutralizing antibodies (bNAbs). While remarkable progress has been made in the isolation and characterization of bNAbs, the epitopes they recognize appear to be poorly immunogenic. Thus, none of the candidate vaccines developed to date has induced satisfactory levels of neutralizing antibodies to the HIV envelope protein (Env). One approach to the problem of poor immunogenicity is to build vaccines based on envelope (env) genes retrieved from rare individuals termed elite neutralizers (ENs) who at one time possessed specific sequences that stimulated the formation of bNAbs. Env proteins selected from these individuals could possess uncommon, yet to be defined, structural features that enhance the immunogenicity of epitopes recognized by bNAbs. Here we describe the recovery of envs from an EN that developed unusually broad and potent bNAbs. As longitudinal specimens were not available, we combined plasma and provirus sequences acquired from a single time-point to infer a phylogenetic tree. Combining ancestral reconstruction data with virus neutralization data allowed us to sift through the myriad of virus quasi-species that evolved in this individual to identify envelope sequences from the nodes that appeared to define the transition from neutralization sensitive envs to the neutralization resistant envs that occur in EN plasma. Synthetic genes from these nodes were functional in infectivity assays and sensitive to neutralization by bNAbs, and may provide a novel source of immunogens for HIV vaccine development., Competing Interests: None of the authors have competing financial or non-financial interests that influenced the collection or interpretation of the data. Although CJP and TW have a commercial affiliation to Monogram Biosciences Inc, this did not alter our adherence to PLoS One policies on sharing data and materials.

Over the last decade, multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein (Env) gp120 have been described. Many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of Envs compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid-containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan-dependent epitopes has been challenging. Here, we report the development of a stable suspension-adapted Chinese hamster ovary (CHO) cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1- CHO cell line exhibit improved binding to prototypic glycan-dependent bN-mAbs directed to the V1/V2 domain (e.g., PG9) and the V3 stem (e.g., PGT128 and 10-1074) while preserving the structure of the important glycan-independent epitopes (e.g., VRC01). The ability of the MGAT1- CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway without impairing the doubling time or ability to grow at high cell densities suggests that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens., Competing Interests: The authors have declared that no competing interests exist.

The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10-100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel robotic cell line selection strategy to address the problems of low expression. Additionally, we employed a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years., Competing Interests: The authors have declared that no competing interests exist.

To date, the RV144 HIV vaccine trial has been the only study to show that immunization can confer protection from HIV infection. While encouraging, the modest 31.2% (P = 0.04) efficacy achieved in this study left significant room for improvement, and created an incentive to optimize the AIDSVAX B/E vaccine immunogens to increase the level of vaccine efficacy. Since the completion of the RV144 trial, our understanding of the antigenic structure of the HIV envelope protein, gp120, and of the specificity of broadly neutralizing monoclonal antibodies (bN-mAbs) that bind to it, has significantly improved. In particular, we have learned that multiple families of bN-mAbs require specific oligomannose glycans for binding. Both of the monomeric gp120 immunogens (MN- and A244-rgp120) in the AIDSVAX B/E vaccine used in the RV144 trial were enriched for glycans containing high levels of sialic acid, and lacked critical N-linked glycosylation sites required for binding by several families of bN-mAbs. The absence of these epitopes may have contributed to the low level of efficacy achieved in this study. In this report, we describe our efforts to improve the antigenic structure of the rgp120 immunogens used in the vaccine by optimizing glycan-dependent epitopes recognized by multiple bN-mAbs. Our results demonstrated that by shifting the location of one PNGS in A244-rgp120, and by adding two PNGS to MN-rgp120, in conjunction with the production of both proteins in a cell line that favors the incorporation of oligomannose glycans, we could significantly improve the binding by three major families of bN-mAbs. The immunogens described here represent a second generation of gp120-based vaccine immunogens that exhibit potential for use in RV144 follow-up studies.

Non-neutralizing IgG to the V1V2 loop of HIV-1 gp120 correlates with a decreased risk of HIV-1 infection but the mechanism of protection remains unknown. This V1V2 IgG correlate was identified in RV144 Thai trial vaccine recipients, who were primed with a canarypox vector expressing membrane-bound gp120 (vCP1521) and boosted with vCP1521 plus a mixture gp120 proteins from clade B and clade CRF01_AE (B/E gp120). We sought to determine whether the mechanism of vaccine protection might involve antibody-dependent complement activation. Complement activation was measured as a function of complement component C3d deposition on V1V2-coated beads in the presence of RV144 sera. Variable levels of complement activation were detected two weeks post final boosting in RV144, which is when the V1V2 IgG correlate was identified. The magnitude of complement activation correlated with V1V2-specific serum IgG and was stronger and more common in RV144 than in HIV-1 infected individuals and two related HIV-1 vaccine trials, VAX003 and VAX004, where no protection was seen. After adjusting for gp120 IgA, V1V2 IgG, gender, and risk score, complement activation by case-control plasmas from RV144 correlated inversely with a reduced risk of HIV-1 infection, with odds ratio for positive versus negative response to TH023-V1V2 0.42 (95% CI 0.18 to 0.99, p = 0.048) and to A244-V1V2 0.49 (95% CI 0.21 to 1.10, p = 0.085). These results suggest that complement activity may have contributed in part to modest protection against the acquisition of HIV-1 infection seen in the RV144 trial.

Background: In the HIV-1 vaccine trial RV144, ALVAC-HIV prime with an AIDSVAX® B/E boost reduced HIV-1 acquisition by 31% at 42 months post first vaccination. The bivalent AIDSVAX® B/E vaccine contains two gp120 envelope glycoproteins, one from the subtype B HIV-1 MN isolate and one from the subtype CRF01_AE A244 isolate. Each envelope glycoprotein harbors a highly conserved 27-amino acid HSV-1 glycoprotein D (gD) tag sequence that shares 93% sequence identity with the HSV-2 gD sequence. We assessed whether vaccine-induced anti-gD antibodies protected females against HSV-2 acquisition in RV144., Methods: Of the women enrolled in RV144, 777 vaccine and 807 placebo recipients were eligible and randomly selected according to their pre-vaccination HSV-1 and HSV-2 serostatus for analysis. Immunoglobulin G (IgG) and IgA responses to gD were determined by a binding antibody multiplex assay and HSV-2 serostatus was determined by Western blot analysis. Ninety-three percent and 75% of the vaccine recipients had anti-gD IgG and IgA responses two weeks post last vaccination, respectively. There was no evidence of reduction in HSV-2 infection by vaccination compared to placebo recipients over 78 weeks of follow-up. The annual incidence of HSV-2 infection in individuals who were HSV-2 negative at baseline or HSV-1 positive and HSV-2 indeterminate at baseline were 4.38/100 person-years (py) and 3.28/100 py in the vaccine and placebo groups, respectively. Baseline HSV-1 status did not affect subsequent HSV-2 acquisition. Specifically, the estimated odds ratio of HSV-2 infection by Week 78 for female placebo recipients who were baseline HSV-1 positive (n = 422) vs. negative (n = 1120) was 1.14 [95% confidence interval 0.66 to 1.94, p = 0.64)]. No evidence of reduction in the incidence of HSV-2 infection by vaccination was detected., Conclusions: AIDSVAX® B/E containing gD did not confer protection from HSV-2 acquisition in HSV-2 seronegative women, despite eliciting anti-gD serum antibodies.

The only evidence of vaccine-induced protection from HIV acquisition in humans was obtained in the RV144 HIV vaccine clinical trial. One immune correlate of risk in RV144 was observed to be higher titers of vaccine-induced antibodies (Abs) reacting with a 23-mer non-glycosylated peptide with the same amino acid sequence as a segment in the second variable (V2) loop of the MN strain of HIV. We used NMR to analyze the dynamic 3D structure of this peptide. Distance restraints between spatially proximate inter-residue protons were calculated from NOE cross peak intensities and used to constrain a thorough search of all possible conformations of the peptide. α-helical folding was strongly preferred by part of the peptide. A high-throughput structure prediction of this segment in all circulating HIV strains demonstrated that α-helical conformations are preferred by this segment almost universally across all subtypes. Notably, α-helical conformations of this segment of the V2 loop cluster cross-subtype-conserved amino acids on one face of the helix and the variable amino acid positions on the other in a semblance of an amphipathic α-helix. Accordingly, some Abs that protected against HIV in RV144 may have targeted a specific, conserved α-helical peptide epitope in the V2 loop of HIV's surface envelope glycoprotein., Competing Interests: The authors have declared that no competing interests exist.

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.

Neutralizing and non-neutralizing antibodies to linear epitopes on HIV-1 envelope glycoproteins have potential to mediate antiviral effector functions that could be beneficial to vaccine-induced protection. Here, plasma IgG responses were assessed in three HIV-1 gp120 vaccine efficacy trials (RV144, Vax003, Vax004) and in HIV-1-infected individuals by using arrays of overlapping peptides spanning the entire consensus gp160 of all major genetic subtypes and circulating recombinant forms (CRFs) of the virus. In RV144, where 31.2% efficacy against HIV-1 infection was seen, dominant responses targeted the C1, V2, V3 and C5 regions of gp120. An analysis of RV144 case-control samples showed that IgG to V2 CRF01_AE significantly inversely correlated with infection risk (OR= 0.54, p=0.0042), as did the response to other V2 subtypes (OR=0.60-0.63, p=0.016-0.025). The response to V3 CRF01_AE also inversely correlated with infection risk but only in vaccine recipients who had lower levels of other antibodies, especially Env-specific plasma IgA (OR=0.49, p=0.007) and neutralizing antibodies (OR=0.5, p=0.008). Responses to C1 and C5 showed no significant correlation with infection risk. In Vax003 and Vax004, where no significant protection was seen, serum IgG responses targeted the same epitopes as in RV144 with the exception of an additional C1 reactivity in Vax003 and infrequent V2 reactivity in Vax004. In HIV-1 infected subjects, dominant responses targeted the V3 and C5 regions of gp120, as well as the immunodominant domain, heptad repeat 1 (HR-1) and membrane proximal external region (MPER) of gp41. These results highlight the presence of several dominant linear B cell epitopes on the HIV-1 envelope glycoproteins. They also generate the hypothesis that IgG to linear epitopes in the V2 and V3 regions of gp120 are part of a complex interplay of immune responses that contributed to protection in RV144.

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.

Background: The 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 Findings: Isoelectric 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/significance: MN- 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.

Background: In 2003, a phase III placebo-controlled trial (VAX003) was completed in Bangkok, Thailand. Of the 2,546 individuals enrolled in the trial based on high risk for infection through injection drug use (IDU), we obtained clinical samples and HIV-1 sequence data (envelope glycoprotein gene gp120) from 215 individuals who became infected during the trial. Here, we used these data in combination with other publicly available gp120 sequences to perform a molecular surveillance and phylodynamic analysis of HIV-1 in Thailand., Methodology and Findings: Phylogenetic and population genetic estimators were used to assess HIV-1 gp120 diversity as a function of vaccination treatment, viral load (VL) and CD4(+) counts, to identify transmission clusters and to investigate the timescale and demographics of HIV-1 in Thailand. Three HIV-1 subtypes were identified: CRF01&#95;AE (85% of the infections), subtype B (13%) and CRF15&#95;AE (2%). The Bangkok IDU cohort showed more gp120 diversity than other Asian IDU cohorts and similar diversity to that observed in sexually infected individuals. Moreover, significant differences (P<0.02) in genetic diversity were observed in CRF01&#95;AE IDU with different VL and CD4(+) counts. No phylogenetic structure was detected regarding any of the epidemiological and clinical factors tested, although high proportions (35% to 50%) of early infections fell into clusters, which suggests that transmission chains associated with acute infection play a key role on HIV-1 spread among IDU. CRF01&#95;AE was estimated to have emerged in Thailand in 1984.5 (1983-1986), 3-6 years before the first recognition of symptomatic patients (1989). The relative genetic diversity of the HIV-1 population has remained high despite decreasing prevalence rates since the mid 1990s., Conclusions: Our study and recent epidemiological reports indicate that HIV-1 is still a major threat in Thailand and suggest that HIV awareness and prevention needs to be strengthened to avoid AIDS resurgence.

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.