Late-Stage Derivatization of Oleanolic Acid-Based Anti-HIV-1 Compounds.

A 12-keto-type oleanolic acid derivative (4) has been identified as a potent anti-human immunodeficiency virus type-1 (HIV-1) compound that demonstrates synergistic effects with several types of HIV-1 neutralizing antibodies. In the present study, we used a common key synthetic intermediate to carry out the late-stage derivatization of an anti-HIV compound based on the chemical structure of a 12-keto-type oleanolic acid derivative. To execute this strategy, we designed a diketo-type oleanolic acid derivative (5) for chemoselective transformation, targeting the carboxy group and the hydroxyl group on the statine unit, as well as the 3-carbonyl group on the oleanolic acid unit, as orthogonal synthetic handles. We carried out four types of chemoselective transformations, leading to identification of the indole-type derivative (16) as a novel potent anti-HIV compound. In addition, further optimization of the ß-hydroxyl group on the statine unit provided the R-4-isobutyl γ-amino acid-type derivative (6), which exhibited potent anti-HIV activity comparable to that of 4 but with reduced cytotoxicity.

Plasmacytoid dendritic cells stimulated with Lactococcus lactis strain Plasma produce soluble factors to suppress SARS-CoV-2 replication.

Innate immune responses are important in the control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. We have previously found a lactic acid bacteria species, Lactococcus lactis strain Plasma (LC-Plasma), which possesses specific feature to activate plasmacytoid dendritic cells (pDCs) and thus may affect innate immune responses. Here, we investigated the impact of pDC activation by LC-Plasma on SARS-CoV-2 replication in vitro. Addition of the culture supernatant of pDCs stimulated with LC-Plasma resulted in suppression of SARS-CoV-2 replication in Vero and Calu-3 cells. We confirmed interferon-α (IFN-α) secretion in the supernatant of pDCs stimulated with LC-Plasma and induction of IFN-stimulated genes in cells treated with the pDC supernatant. Anti-IFN-α antibody impaired the suppression of SARS-CoV-2 replication by the supernatant of LC-Plasma-stimulated pDCs, suggesting that IFN-α plays an important role in the SARS-CoV-2 suppression. Our results indicate the potential of LC-Plasma to induce inhibitory responses against SARS-CoV-2 replication through pDC stimulation with IFN-α secretion.

Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques.

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure.

Development of a novel Macaque-Tropic HIV-1 adapted to cynomolgus macaques.

Macaque-tropic HIV-1 (HIV-1mt) variants have been developed to establish preferable primate models that are advantageous in understanding HIV-1 infection pathogenesis and in assessing the preclinical efficacy of novel prevention/treatment strategies. We previously reported that a CXCR4-tropic HIV-1mt, MN4Rh-3, efficiently replicates in peripheral blood mononuclear cells (PBMCs) of cynomolgus macaques homozygous for TRIMCyp (CMsTC). However, the CMsTC challenged with MN4Rh-3 displayed low viral loads during the acute infection phase and subsequently exhibited short-term viremia. These virological phenotypes in vivo differed from those observed in most HIV-1-infected people. Therefore, further development of the HIV-1mt variant was needed. In this study, we first reconstructed the MN4Rh-3 clone to produce a CCR5-tropic HIV-1mt, AS38. In addition, serial in vivo passages allowed us to produce a highly adapted AS38-derived virus that exhibits high viral loads (up to approximately 106 copies ml-1) during the acute infection phase and prolonged periods of persistent viremia (lasting approximately 16 weeks postinfection) upon infection of CMsTC. Whole-genome sequencing of the viral genomes demonstrated that the emergence of a unique 15-nt deletion within the vif gene was associated with in vivo adaptation. The deletion resulted in a significant increase in Vpr protein expression but did not affect Vif-mediated antagonism of antiretroviral APOBEC3s, suggesting that Vpr is important for HIV-1mt adaptation to CMsTC. In summary, we developed a novel CCR5-tropic HIV-1mt that can induce high peak viral loads and long-term viremia and exhibits increased Vpr expression in CMsTC.

Design, synthesis, and bio-evaluation of novel triterpenoid derivatives as anti-HIV-1 compounds.

Two betulinic acid derivatives, RPR103611 (2) and IC9564 (3) were previously reported to be potent HIV-1 entry inhibitors. In this current study, a SAR study of the triterpenoid moiety of 2 and 3 has been performed and an oleanolic acid derivative (4) was identified as a novel HIV-1 entry inhibitor. In addition, the combination of 4 with several-type of HIV-1 neutralizing antibodies provided significant synergistic effects. The synthetic utility of the CC double bond in the C-ring of 4 was also demonstrated to develop the 12-keto-type oleanolic acid derivative (5) as a potent anti-HIV compound. This simple transformation led to a significantly increased anti-HIV activity and a reduced cytotoxicity of the compound.

Hybrids of small CD4 mimics and gp41-related peptides as dual-target HIV entry inhibitors.

Hybrid molecules containing small CD4 mimics and gp41-C-terminal heptad repeat (CHR)-related peptides have been developed. A YIR-821 derivative was adopted as a CD4 mimic, which inhibits the interaction of gp120 with CD4. SC-peptides, SC34 and SC22EK, were also used as CHR-related peptides, which inhibit the interaction between the N-terminal heptad repeat (NHR) and CHR and thereby membrane fusion. Therefore, these hybrid molecules have dual-targets of gp120 and gp41. In the synthesis of the hybrid molecules of CD4 mimic-SC-peptides with different lengths of linkers, two conjugating methods, Cu-catalyzed azide-alkyne cycloaddition and direct cysteine alkylation, were performed. The latter reaction caused simpler operation procedures and higher synthetic yields than the former. The synthesized hybrid molecules of CD4 mimic-SC22EK have significantly higher anti-HIV activity than each sole agent. The present data should be useful in the future design of anti-HIV agents as dual-target entry inhibitors.

Exploratory studies on soluble small molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics, which inhibit the interaction of gp120 with CD4, have been developed. Original CD4 mimics such as NBD-556, which has an aromatic ring, an oxalamide linker and a piperidine moiety, possess significant anti-HIV activity but with their hydrophobic aromatic ring-containing structures are poorly soluble in water. We have developed derivatives with a halopyridinyl group in place of the phenyl group, such as KKN-134, and found them to have excellent aqueous solubility. Other leads that were examined are YIR-821, a compound with a cyclohexane group in a spiro attachment to a piperidine ring and a guanidino group on the piperidine nitrogen atom, and its PEGylated derivative, TKB-002. YIR-821 and TKB-002 retain potent anti-HIV activity. Here, new CD4 mimics, in which the phenyl group was replaced by a halopyridinyl group with the halogen atoms in different positions, their derivatives without a cyclohexane group on the piperidine ring and their hybrid molecules with PEG units were designed and synthesized. Some of these compounds show significantly higher aqueous solubility with maintenance of certain levels of anti-HIV activity. The present data should be useful in the future design of CD4 mimic molecules.

Soluble-type small-molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics have been reported previously as HIV-1 entry inhibitors, which block the interaction between the Phe43 cavity of HIV-1 gp120 and the host CD4. Known CD4 mimics such as NBD-556 possess significant anti-HIV activity but are less soluble in water, perhaps due to their hydrophobic aromatic ring-containing structures. Compounds with a pyridinyl group in place of the phenyl group in these molecules have been designed and synthesized in an attempt to increase the hydrophilicity. Some of these new CD4 mimics, containing a tetramethylpiperidine ring show significantly higher water solubility than NBD-556 and have high anti-HIV activity and synergistic anti-HIV activity with a neutralizing antibody. The CD4 mimic that has a cyclohexylpiperidine ring and a 6-fluoropyridin-3-yl ring has high anti-HIV activity and no significant cytotoxicity. The present results will be useful in the future design and development of novel soluble-type molecule CD4 mimics.

Isolation and structure determination of a new lasso peptide specialicin based on genome mining.

Based on genome mining, a new lasso peptide specialicin was isolated from the extract of Streptomyces specialis. The structure of specialicin was established by ESI-MS and NMR analyses to be a lasso peptide with the length of 21 amino acids, containing an isopeptide bond and two disulfide bonds in the molecule. The stereochemistries of the constituent amino acids except for Trp were determined to be L and the stereochemistry of Trp at C-terminus was determined to be D. Three dimensional structure of specialicin was determined based on NOE experimental data, which indicated that specialicin possessed the similar conformational structure with siamycin I. Specialicin showed the antibacterial activity against Micrococcus luteus and the moderate anti-HIV activity against HIV-1 NL4-3. The biosynthetic gene cluster of specialicin was proposed from the genome sequence data of S. specialis.

Flexibility of small molecular CD4 mimics as HIV entry inhibitors.

CD4 mimics such as YIR-821 and its derivatives are small molecules which inhibit the interaction between the Phe43 cavity of HIV-1 gp120 with host CD4, an interaction that is involved in the entry of HIV to cells. Known CD4 mimics generally possess three structural features, an aromatic ring, an oxalamide linker and a piperidine moiety. We have shown previously that introduction of a cyclohexyl group and a guanidine group into the piperidine moiety and a fluorine atom at the meta-position of the aromatic ring leads to a significant increase in the anti-HIV activity. In the current study, the effects of conformational flexibility were investigated by introduction of an indole-type group in the junction between the oxalamide linker and the aromatic moiety or by replacement of the oxalamide linker with a glycine linker. This led to the development of compounds with high anti-HIV activity, showing the importance of the junction region for the expression of high anti-HIV activity. The present data are expected to be useful in the future design of novel CD4 mimic molecules.

Biphasic CD8+ T-Cell Defense in Simian Immunodeficiency Virus Control by Acute-Phase Passive Neutralizing Antibody Immunization.

UNLABELLED: Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of postinfection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here, we describe SIV control in macaques showing biphasic CD8(+) cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIVmac239-infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8(+) cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second, sustained phase of SIV control, CTL responses converged on a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK)(hi)/phosphorylated AMP-activated protein kinase (AMPK)(lo) subpopulations, implying their correlation with SIV control. The results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control. IMPORTANCE: In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence, it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs with CTL cross-priming capacity resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control, and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection.

Fibrocytes Differ from Macrophages but Can Be Infected with HIV-1.

Fibrocytes (fibroblastic leukocytes) are recently identified as unique hematopoietic cells with features of both macrophages and fibroblasts. Fibrocytes are known to contribute to the remodeling or fibrosis of various injured tissues. However, their role in viral infection is not fully understood. In this study, we show that differentiated fibrocytes are phenotypically distinguishable from macrophages but can be infected with HIV-1. Importantly, fibrocytes exhibited persistently infected cell-like phenotypes, the degree of which was more apparent than macrophages. The infected fibrocytes produced replication-competent HIV-1, but expressed HIV-1 mRNA at low levels and strongly resisted HIV-1-induced cell death, which enabled them to support an extremely long-term HIV-1 production at low but steady levels. More importantly, our results suggested that fibrocytes were susceptible to HIV-1 regardless of their differentiation state, in contrast to the fact that monocytes become susceptible to HIV-1 after the differentiation into macrophages. Our findings indicate that fibrocytes are the previously unreported HIV-1 host cells, and they suggest the importance of considering fibrocytes as one of the long-lived persistently infected cells for curing HIV-1.

[Clinical Significance of Circulating Tumor DNA and Its Future Prospects].

"Liquid biopsy"is a diagnostic medium to detect very small amount of tumor-derived molecules in blood for selecting precise clinical treatments and giving a diagnosis. This article focuses on circulating tumor DNA(ctDNA)and describes its features, clinical significance as well as future prospects with recent findings in this field. "Liquid biopsy"using ctDNAis facilitated by recent developments of highly sensitive technologies, including digital PCR. "BEAMing"is a digital PCR method that can detect rare mutations in ctDNAand will be introduced with some clinical evidences in this article. ctDNAis tumorderived fragmented DNAthat is circulating freely in the blood of a cancer patient. Tests for ctDNAare less invasive, easier than tissue-based tests to collect specimens and can be used repeatedly regardless of any cancer types. Emerging clinical evidences indicate that ctDNAtests show a high concordance rate with tissue-based tests in the identification of oncogene genotypes and can be used for real-time monitoring in vivo, such as prediction of clinical treatments, recurrence and drug resistance. "Liquid biopsy" using ctDNAcontinues to develop, but is promising to complement conventional tissue-based tests. Clinical applications of ctDNAtests are expected to expand as a useful diagnostic tools in areas of therapy selection and monitoring for drug resistance.

A minimally cytotoxic CD4 mimic as an HIV entry inhibitor.

Several CD4 mimics have been reported as HIV-1 entry inhibitors which can block the interaction between the viral envelope glycoprotein gp120 and the cell surface protein CD4. We previously found a lead compound 2 (YYA-021) with high anti-HIV activity and low cytotoxicity. Pharmacokinetic analysis however showed compound 2 to have wide tissue distribution and relatively high distribution volumes in rats and rhesus macaques. In the present study we searched for more hydrophilic CD4 mimics with a view to reducing tissue distribution. A new compound (5) with a 1,3-benzodioxolyl moiety was found to have relatively high anti-HIV activity and no significant cytotoxicity. Compound 5 is more hydrophilic than compound 2 and the pharmacokinetics of the intravenous administration of compound 5 in a rhesus macaque showed that compound 5 has lower tissue distribution than compound 2, suggesting that compound 5 possesses a better profile.

Impact of maraviroc-resistant and low-CCR5-adapted mutations induced by in vitro passage on sensitivity to anti-envelope neutralizing antibodies.

The aim of this study was to generate maraviroc (MVC)-resistant viruses in vitro using a human immunodeficiency virus type 1 subtype B clinical isolate (HIV-1KP-5) to understand the mechanism(s) of resistance to MVC. To select HIV-1 variants resistant to MVC in vitro, we exposed high-chemokine (C-C motif) receptor 5 (CCR5)-expressing PM1/CCR5 cells to HIV-1KP-5 followed by serial passage in the presence of MVC. We also passaged HIV-1KP-5 in PM1 cells, which were low CCR5 expressing to determine low-CCR5-adapted substitutions and compared the Env sequences of the MVC-selected variants. Following 48 passages with MVC (10 µM), HIV-1KP-5 acquired a resistant phenotype [maximal per cent inhibition (MPI) 24%], whilst the low-CCR5-adapted variant had low sensitivity to MVC (IC50 ~200 nM), but not reduction of the MPI. The common substitutions observed in both the MVC-selected and low-CCR5-adapted variants were selected from the quasi-species, in V1, V3 and V5. After 14 passages, the MVC-selected variants harboured substitutions around the CCR5 N-terminal-binding site and V3 (V200I, T297I, K305R and M434I). The low-CCR5-adapted infectious clone became sensitive to anti-CD4bs and CD4i mAbs, but not to anti-V3 mAb and autologous plasma IgGs. Conversely, the MVC-selected clone became highly sensitive to the anti-envelope (Env) mAbs tested and the autologous plasma IgGs. These findings suggest that the four MVC-resistant mutations required for entry using MVC-bound CCR5 result in a conformational change of Env that is associated with a phenotype sensitive to anti-Env neutralizing antibodies.

Mpox Neutralizing Antibody Response to LC16m8 Vaccine in Healthy Adults.

BACKGROUND: Vaccination against mpox (formerly known as monkeypox), an infectious disease caused by the monkeypox virus (MPXV), is needed to prevent outbreaks and consequent public health concerns. The LC16m8 vaccine, a dried cell-cultured proliferative live attenuated vaccinia virus­based vaccine, was approved in Japan against smallpox and mpox. However, its immunogenicity and efficacy against MPXV have not been fully assessed. We assessed the safety and immunogenicity of LC16m8 against MPXV in healthy adults. METHODS: We conducted a single-arm study that included 50 participants who were followed up for 168 days postvaccination. The primary end point was the neutralizing antibody seroconversion rate against MPXVs, including the Zr599 and Liberia strains, on day 28. The secondary end points included the vaccine "take" (major cutaneous reaction) rate, neutralizing titer kinetics against MPXV and vaccinia virus (LC16m8) strains, and safety outcomes. RESULTS: Seroconversion rates on day 28 were 72% (36 of 50), 70% (35 of 50), and 88% (44 of 50) against the Zr599 strain, the Liberia strain, and LC16m8, respectively. On day 168, seroconversion rates decreased to 30% (15 of 50) against the Zr599 and Liberia strains and to 76% (38 of 50) against LC16m8. The vaccine "take" (broad definition) rate on day 14 was 94% (46 of 49). Adverse events (AEs), including common solicited cutaneous reactions, occurred in 98% (45 of 48) of participants; grade 3 severity AEs occurred in 16% (8 of 50). No deaths, serious AEs, or mpox onset incidences were observed up to day 168. CONCLUSIONS: The LC16m8 vaccine generated neutralizing antibody responses against MPXV in healthy adults. No serious safety concerns occurred with LC16m8 use. (Funded by the Ministry of Health, Labour and Welfare of Japan; Japan Registry of Clinical Trials number, jRCTs031220171.)

Impact of antiretroviral pressure on selection of primary human immunodeficiency virus type 1 envelope sequences in vitro.

The initiation of drug therapy results in a reduction in the human immunodeficiency virus type 1 (HIV-1) population, which represents a potential genetic bottleneck. The effect of this drug-induced genetic bottleneck on the population dynamics of the envelope (Env) regions has been addressed in several in vivo studies. However, it is difficult to investigate the effect on the env gene of the genetic bottleneck induced not only by entry inhibitors but also by non-entry inhibitors, particularly in vivo. Therefore, this study used an in vitro selection system using unique bulk primary isolates established in the laboratory to observe the effects of the antiretroviral drug-induced bottleneck on the integrase and env genes. Env diversity was decreased significantly in one primary isolate [KP-1, harbouring both CXCR4 (X4)- and CCR5 (R5)-tropic variants] when passaged in the presence or absence of raltegravir (RAL) during in vitro selection. Furthermore, the RAL-selected KP-1 variant had a completely different Env sequence from that in the passage control (particularly evident in the gp120, V1/V2 and V4-loop regions), and a different number of potential N-glycosylation sites. A similar pattern was also observed in other primary isolates when using different classes of drugs. This is the first study to explore the influence of anti-HIV drugs on bottlenecks in bulk primary HIV isolates with highly diverse Env sequences using in vitro selection.

CD4 mimics as HIV entry inhibitors: lead optimization studies of the aromatic substituents.

Several CD4 mimics have been reported as HIV-1 entry inhibitors that can intervene in the interaction between a viral envelope glycoprotein gp120 and a cell surface protein CD4. Our previous SAR studies led to a finding of a highly potent analogue 3 with bulky hydrophobic groups on a piperidine moiety. In the present study, the aromatic ring of 3 was modified systematically in an attempt to improve its antiviral activity and CD4 mimicry which induces the conformational changes in gp120 that can render the envelope more sensitive to neutralizing antibodies. Biological assays of the synthetic compounds revealed that the introduction of a fluorine group as a meta-substituent of the aromatic ring caused an increase of anti-HIV activity and an enhancement of a CD4 mimicry, and led to a novel compound 13a that showed twice as potent anti-HIV activity compared to 3 and a substantial increase in a CD4 mimicry even at lower concentrations.

A CD4 mimic as an HIV entry inhibitor: pharmacokinetics.

To date, several small molecules of CD4 mimics, which can suppress competitively the interaction between an HIV-1 envelope glycoprotein gp120 and a cellular surface protein CD4, have been reported as viral entry inhibitors. A lead compound 2 (YYA-021) with relatively high potency and low cytotoxicity has been identified previously by SAR studies. In the present study, the pharmacokinetics of the intravenous administration of compound 2 in rats and rhesus macaques is reported. The half-lives of compound 2 in blood in rats and rhesus macaques suggest that compound 2 shows wide tissue distribution and relatively high distribution volumes. A few hours after the injection, both plasma concentrations of compound 2 maintained micromolar levels, indicating it might have promise for intravenous administration when used combinatorially with anti-gp120 monoclonal antibodies.

Association of envelope-specific B-cell differentiation and viral selective pressure signatures in HIV-1 CRF01_AE infection.

OBJECTIVE: In HIV type 1 (HIV-1) infection, virus-specific B-cell and neutralizing antibody (NAb) responses are impaired but exert selective pressure on target viral Envelope (Env) resulting in prominent sequence diversification among geographical areas. The basal induction patterns of HIV Env-specific B cells and their interaction with HIV Env awaits clarification. DESIGN: We investigated the relationship of Env polymorphisms and Env-specific B-cell responses in treatment-naive HIV-1 CRF01_AE-infected Vietnamese. METHODS: Samples of 43 HIV-1 CRF01_AE infection-identified individuals were divided into acute-phase ( n  = 12) and chronic-phase ( n  = 31) by combined criteria of serological recent-infection assay and clinical parameters. We quantified subcloning-based polymorphic residue site numbers in plasma-derived Env variable region 1-5 (V1-V5)-coding regions within each individual, designating their summation within each region as variant index. Peripheral blood Env gp 140-specific B-cell responses and plasma neutralizing activity of Env pseudoviruses were examined to analyze their relationship with variant index. RESULTS: HIV-1 CRF01_AE Env gp140-specific total B-cell and plasma cell (CD19 + IgD - CD27 + CD38 + CD138 + ) responses were determined. In chronic-phase samples, significant correlation of variant index in all Env V1-V5 regions with Env-specific plasma cell responses was shown, and V1-V5 total variant index correlated stronger with Env-specific plasma cell as compared with total Env-specific B-cell responses. Env V5 variant index was significantly higher in chronic-phase cross-neutralizers of V5-polymorphic/VRC01-insensitive CRF01_AE Env. CONCLUSION: Results revealed the association between circulating Env-specific plasma cell responses and Env polymorphisms, implicating selective pressure on Env by plasma cell-derived antibodies and conversely suggests that Env-specific B-cell induction alone is insufficient for exerting Env selective pressure in HIV infection.