Factors Associated with Neutralizing Antibody Responses following 2-Dose and 3rd Booster Monovalent COVID-19 Vaccination in Japanese People Living with HIV.

People living with HIV (PLWH) could be at risk of blunted immune responses to COVID-19 vaccination. We investigated factors associated with neutralizing antibody (NAb) responses against SARS-CoV-2 and variants of concern (VOCs), following two-dose and third booster monovalent COVID-19 mRNA vaccination in Japanese PLWH. NAb titers were assessed in polyclonal IgG fractions by lentiviral-based pseudovirus assays. Overall, NAb titers against Wuhan, following two-dose vaccination, were assessed in 82 PLWH on treatment, whereby 17/82 (20.73%) were classified as low-NAb participants. Within the low-NAb participants, the third booster vaccination enhanced NAb titers against Wuhan and VOCs, albeit to a significantly lower magnitude than the rest. In the multivariate analysis, NAb titers against Wuhan after two-dose vaccination correlated with age and days since vaccination, but not with CD4+ count, CD4+/CD8+ ratio, and plasma high-sensitivity C-Reactive protein (hsCRP). Interestingly, an extended analysis within age subgroups revealed NAb titers to correlate positively with the CD4+ count and negatively with plasma hsCRP in younger, but not older, participants. In conclusion, a third booster vaccination substantially enhances NAb titers, but the benefit may be suboptimal in subpopulations of PLWH exhibiting low titers at baseline. Considering clinical and immune parameters could provide a nuanced understanding of factors associated with vaccine responses in PLWH.

Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19) and employs angiotensin-converting enzyme 2 (ACE2) as the receptor. Although the expression of ACE2 is crucial for cellular entry, we found that the interaction between ACE2 and the Spike (S) protein in the same cells led to its downregulation through degradation in the lysosomal compartment via the endocytic pathway. Interestingly, the ability of the S protein from previous variants of concern (VOCs) to downregulate ACE2 was variant-dependent and correlated with disease severity. The S protein from the Omicron variant, associated with milder disease, exhibited a lower capacity to downregulate ACE2 than that of the Delta variant, which is linked to a higher risk of hospitalization. Chimeric studies between the S proteins from the Delta and Omicron variants revealed that both the receptor-binding domain (RBD) and the S2 subunit played crucial roles in the reduced ACE2 downregulation activity observed in the Omicron variant. In contrast, three mutations (L452R/P681R/D950N) located in the RBD, S1/S2 cleavage site, and HR1 domain were identified as essential for the higher ACE2 downregulation activity observed in the Delta variant compared to that in the other VOCs. Our results suggested that dysregulation of the renin-angiotensin system due to the ACE2 downregulation activity of the S protein of SARS-CoV-2 may play a key role in the pathogenesis of COVID-19.

Idiotopes of antibodies against HIV-1 CD4-induced epitope shared with those against microorganisms.

Induction of antibodies (Abs) against the conformational CD4-induced (CD4i) epitope is frequent in HIV-1 infection. However, the mechanism of development of anti-CD4i Abs is unclear. We used anti-idiotypic (aID) monoclonal Abs (mAbs) of anti-CD4i mAbs to isolate anti-CD4i mAbs from infected subjects and track the causative antigens. One anti-aID mAb sorted from infected subjects by aID mAbs had the characteristics of anti-CD4i Abs, including IGHV1-69 usage and ability to bind to HIV-1 Env enhanced by sCD4. Critical amino acid sequences for the binding of six anti-aID mAbs, with shared idiotope to anti-CD4i mAbs, were analysed by phage display. The identified amino acid sequences showed similarity to proteins from human microbiota and infectious agents. Peptides synthesized from Caudoviricetes sp and Vibrio vulnificus based on the identified sequences were reactive to most anti-aID and some anti-CD4i mAbs. These results suggest that anti-CD4i Abs may evolve from B cells primed by microorganisms.

Evaluation of Neutralizing Activity against Omicron Subvariants in BA.5 Breakthrough Infection and Three-Dose Vaccination Using a Novel Chemiluminescence-Based, Virus-Mediated Cytopathic Assay.

Neutralizing potency of humoral immune responses induced by prior infection or vaccination is vital for protecting of individuals and population against severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). However, the emergence of viral variants that can evade neutralization by vaccine- or infection-induced immunity is a significant public health threat and requires continuous monitoring. Here, we have developed a novel scalable chemiluminescence-based assay for assessing SARS-CoV-2-induced cytopathic effect to quantify the neutralizing activity of antisera. The assay leverages the correlation between host cell viability and ATP levels in culture to measure the cytopathic effect on target cells induced by clinically isolated, replication-competent, authentic SARS-CoV-2. With this assay, we demonstrate that the recently arisen Omicron subvariants BQ.1.1 and XBB.1 display a significant decrease in sensitivity to neutralization by antibodies elicited from breakthrough infections with Omicron BA.5 and from receipt of three doses of mRNA vaccines. Thus, this scalable neutralizing assay provides a useful platform to assess the potency of acquired humoral immunity against newly emerging SARS-CoV-2 variants. IMPORTANCE The ongoing global pandemic of SARS-CoV-2 has emphasized the importance of neutralizing immunity in protecting individuals and populations against severe respiratory illness. In light of the emergence of viral variants with the potential to evade immunity, continuous monitoring is imperative. A virus plaque reduction neutralization test (PRNT) is a "gold standard" assay for analyzing neutralizing activity for authentic viruses that form plaques, like influenza virus, dengue virus, and SARS-CoV-2. However, this method is labor intensive and is not efficient for performing large-scale neutralization assays on patient specimens. The assay system established in this study allows for the detection of a patient's neutralizing activity by simply adding an ATP detection reagent, providing a simple evaluation system for neutralizing activity of antisera as an alternative to the plaque reduction method. Our extended analysis of the Omicron subvariants highlights their increasing capability to evade neutralization by both vaccine- and infection-induced humoral immunity.

Characterization of a Novel CD4 Mimetic Compound YIR-821 against HIV-1 Clinical Isolates.

Small CD4-mimetic compound (CD4mc), which inhibits the interaction between gp120 with CD4, acts as an entry inhibitor and induces structural changes in the HIV-1 envelope glycoprotein trimer (Env) through its insertion within the Phe43 cavity of gp120. We recently developed YIR-821, a novel CD4mc, that has potent antiviral activity and lower toxicity than the prototype NBD-556. To assess the possibility of clinical application of YIR-821, we tested its antiviral activity using a panel of HIV-1 pseudoviruses from different subtypes. YIR-821 displayed entry inhibitor activity against 53.5% (21/40) of the pseudoviruses tested and enhanced neutralization mediated by coreceptor binding site (CoRBS) antibodies in 50% (16/32) of these. Furthermore, when we assessed the antiviral effects using a panel of pseudoviruses and autologous plasma IgG, enhancement of antibody-mediated neutralization activity was observed for 48% (15/31) of subtype B strains and 51% (28/55) of non-B strains. The direct antiviral activity of YIR-821 as an entry inhibitor was observed in 53% of both subtype B (27/51) and non-B subtype (40/75) pseudoviruses. Enhancement of antibody-dependent cellular cytotoxicity was also observed with YIR-821 for all six selected clinical isolates, as well as for the transmitted/founder (T/F) CH58 virus-infected cells. The sequence diversity in the CD4 binding site as well as other regions, such as the gp120 inner domain layers or gp41, may be involved in the multiple mechanisms related to the sensitive/resistant phenotype of the virus to YIR-821. Our findings may facilitate the clinical application of YIR-821. IMPORTANCE Small CD4-mimetic compound (CD4mc) interacts with the Phe43 cavity and triggers conformational changes, enhancing antibody-mediated neutralization and antibody-dependent cellular cytotoxicity (ADCC). Here, we evaluated the effect of YIR-821, a novel CD4mc, against clinical isolates, including both subtype B and non-B subtype viruses. Our results confirm the desirable properties of YIR-821, which include entry inhibition, enhancement of IgG-neutralization, binding, and ADCC, in addition to low toxicity and long half-life in a rhesus macaque model, that might facilitate the clinical application of this novel CD4mc. Our observation of primary viruses that are resistant to YIR-821 suggests that further development of CD4mcs with different structural properties is required.

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.

Functional analysis of a monoclonal antibody reactive against the C1C2 of Env obtained from a patient infected with HIV-1 CRF02_AG.

BACKGROUND: Recent data suggest the importance of non-neutralizing antibodies (nnAbs) in the development of vaccines against HIV-1 because two types of nnAbs that recognize the coreceptor binding site (CoRBS) and the C1C2 region mediate antibody-dependent cellular-cytotoxicity (ADCC) against HIV-1-infected cells. However, many studies have been conducted with nnAbs obtained from subtype B-infected individuals, with few studies in patients with non-subtype B infections. RESULTS: We isolated a monoclonal antibody 1E5 from a CRF02_AG-infected individual and constructed two forms of antibody with constant regions of IgG1 or IgG3. The epitope of 1E5 belongs to the C1C2 of gp120, and 1E5 binds to 27 out of 35 strains (77 %) across the subtypes. The 1E5 showed strong ADCC activity, especially in the form of IgG3 in the presence of small CD4-mimetic compounds (CD4mc) and 4E9C (anti-CoRBS antibody), but did not show any neutralizing activity even against the isolates with strong binding activities. The enhancement in the binding of A32, anti-C1C2 antibody isolated from a patient with subtype B infection, was observed in the presence of 1E5 and the combination of 1E5, A32 and 4E9C mediated a strong ADCC activity. CONCLUSIONS: These results suggest that anti-C1C2 antibodies that are induced in patients with different HIV-1 subtype infections have common functional modality and may have unexpected interactions. These data may have implications for vaccine development against HIV-1.

Neutralizing Antibody Induction Associated with a Germline Immunoglobulin Gene Polymorphism in Neutralization-Resistant SIVsmE543-3 Infection.

Antibody responses are crucial for the control of virus infection. Understanding of the mechanism of antibody induction is important for the development of a vaccine eliciting effective anti-virus antibodies. Virus-specific B cell receptor (BCR)/antibody repertoires are different among individuals, but determinants for this difference remain largely unclear. We have recently reported that a germline BCR immunoglobulin (IgG) gene polymorphism (VH3.33_ET or VH3.33_VI) in rhesus macaques is the determinant for induction of potent B404-class anti-simian immunodeficiency virus (SIV) neutralizing antibodies in neutralization-sensitive SIVsmH635FC infection. In the present study, we examined whether neutralization-resistant SIVsmE543-3 infection can induce the anti-SIV neutralizing antibodies associated with the germline VH3.33 polymorphism. Anti-SIVsmE543-3 neutralizing antibodies were induced in all the macaques possessing the VH3.33_ET allele, but not in those without VH3.33_ET, in the chronic phase of SIVsmE543-3 infection. Next generation sequencing analysis of BCR VH genes found B404-class antibody sequences only in those with VH3.33_ET. These results indicate that anti-SIVsmE543-3 neutralizing antibody induction associated with the germline BCR IgG gene polymorphism can be triggered by infection with neutralization-resistant SIVsmE543-3. This animal model would be useful for the elucidation of the mechanism of potent antibody induction against neutralization-resistant viruses.

Resistance of SARS-CoV-2 variants to neutralization by antibodies induced in convalescent patients with COVID-19.

Administration of convalescent plasma or neutralizing monoclonal antibodies (mAbs) is a potent therapeutic option for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, SARS-CoV-2 variants with mutations in the spike protein have emerged in many countries. To evaluate the efficacy of neutralizing antibodies induced in convalescent patients against emerging variants, we isolate anti-spike mAbs from two convalescent COVID-19 patients infected with prototypic SARS-CoV-2 by single-cell sorting of immunoglobulin-G-positive (IgG+) memory B cells. Anti-spike antibody induction is robust in these patients, and five mAbs have potent neutralizing activities. The efficacy of most neutralizing mAbs and convalescent plasma samples is maintained against B.1.1.7 and mink cluster 5 variants but is significantly decreased against variants B.1.351 from South Africa and P.1 from Brazil. However, mAbs with a high affinity for the receptor-binding domain remain effective against these neutralization-resistant variants. Rapid spread of these variants significantly impacts antibody-based therapies and vaccine strategies against SARS-CoV-2.

Phenotypic and Genotypic Co-receptor Tropism Testing in HIV-1 Epidemic Region of Tanzania Where Multiple Non-B Subtypes Co-circulate.

HIV human immunodeficiency virus type I (HIV-1) entry inhibitor potency is dependent on viral co-receptor tropisms and thereby tropism determination is clinically important. However, phenotypic tropisms of HIV-1 non-B subtypes have been poorly investigated and the genotypic prediction algorithms remain insufficiently validated. To clarify this issue, we recruited 52 treatment-naïve, HIV-1-infected patients in Tanzania, where multiple HIV-1 non-B subtypes co-circulate. Sequence analysis of 93 infectious envelope clones isolated from their plasma viral RNA revealed the co-circulation of subtypes A1, C, D, and inter-subtype recombinant forms (isRFs). Phenotypic tropism assays revealed that lentivirus reporters pseudotyped with 75 (80.6%) and 5 (5.4%) envelope clones could establish infection toward U87.CD4 cells expressing CCR5 (R5) and CXCR4 (X4), respectively; whereas the remaining 13 (14%) clones could infect both cells. Genotypic analyses by widely used algorithms including V3 net charge, Geno2pheno, WebPSSM, and PhenoSeq showed that almost all phenotypic X4-tropic clones and only 15 of 75 phenotypic R5-tropic clones were concordantly predicted. However, the remaining 60 phenotypic R5-tropic clones were discordantly predicted by at least one algorithm. In particular, 2 phenotypic R5-tropic clones were discordantly predicted by all algorithms tested. Taken together, the results demonstrate the limitation of currently available genotypic algorithms for predicting co-receptor inference among co-circulating multiple non-B subtypes and emerging isRFs. Also, the phenotypic tropism dataset presented here could be valuable for retraining of the widely used genotypic prediction algorithms to enhance their performance.

A Potent anti-Simian Immunodeficiency Virus Neutralizing Antibody Induction Associated with a Germline Immunoglobulin Gene Polymorphism in Rhesus Macaques.

Virus infection induces B cells with a wide variety of B cell receptor (BCR) repertoires. Patterns of induced BCR repertoires are different in individuals, while the underlying mechanism causing this difference remains largely unclear. In particular, the impact of germ line BCR immunoglobulin (Ig) gene polymorphism on B cell/antibody induction has not fully been determined. In the present study, we found a potent antibody induction associated with a germ line BCR Ig gene polymorphism. B404-class antibodies, which were previously reported as potent anti-simian immunodeficiency virus (SIV) neutralizing antibodies using the germ line VH3.33 gene-derived Ig heavy chain, were induced in five of 10 rhesus macaques after SIVsmH635FC infection. Investigation of VH3.33 genes in B404-class antibody inducers (n = 5) and non-inducers (n = 5) revealed association of B404-class antibody induction with a germ line VH3.33 polymorphism. Analysis of reconstructed antibodies indicated that the VH3.33 residue 38 is the determinant for B404-class antibody induction. B404-class antibodies were induced in all the macaques possessing the B404-associated VH3.33 allele, even under undetectable viremia. Our results show that a single nucleotide polymorphism in germ line VH genes could be a determinant for induction of potent antibodies against virus infection, implying that germ line VH-gene polymorphisms can be a factor restricting effective antibody induction or responsiveness to vaccination.IMPORTANCE Vaccines against a wide variety of infectious diseases have been developed mostly to induce antibodies targeting pathogens. However, small but significant percentage of people fail to mount potent antibody responses after vaccination, while the underlying mechanism of host failure in antibody induction remains largely unclear. In particular, the impact of germ line B cell receptor (BCR)/antibody immunoglobulin (Ig) gene polymorphism on B cell/antibody induction has not fully been determined. In the present study, we found a potent anti-simian immunodeficiency virus neutralizing antibody induction associated with a germ line BCR/antibody Ig gene polymorphism in rhesus macaques. Our results demonstrate that a single nucleotide polymorphism in germ line Ig genes could be a determinant for induction of potent antibodies against virus infection, implying that germ line BCR/antibody Ig gene polymorphisms can be a factor restricting effective antibody induction or responsiveness to vaccination.

Hybrids of Small-Molecule CD4 Mimics with Polyethylene Glycol Units as HIV Entry Inhibitors.

CD4 mimics are small molecules that inhibit the interaction of gp120 with CD4. We have developed several CD4 mimics. Herein, hybrid molecules consisting of CD4 mimics with a long alkyl chain or a PEG unit attached through a self-cleavable linker were synthesized. In anti-HIV activity, modification with a PEG unit appeared to be more suitable than modification with a long alkyl chain. Thus, hybrid molecules of CD4 mimics, with PEG units attached through an uncleavable linker, were developed and showed high anti-HIV activity and low cytotoxicity. In investigation of pharmacokinetics in a rhesus macaque, a hybrid compound had a more effective PK profile than that of the parent compound, and intramuscular injection was a more useful administration route to maintain the high blood concentration of the CD4 mimic than intravenous injection. The presented hybrid molecules of CD4 mimics with a PEG unit would be practically useful when combined with a neutralizing antibody.

Existence of Replication-Competent Minor Variants with Different Coreceptor Usage in Plasma from HIV-1-Infected Individuals.

Cell entry by HIV-1 is mediated by its principal receptor, CD4, and a coreceptor, either CCR5 or CXCR4, with viral envelope glycoprotein gp120. Generally, CCR5-using HIV-1 variants, called R5, predominate over most of the course of infection, while CXCR4-using HIV-1 variants (variants that utilize both CCR5 and CXCR4 [R5X4, or dual] or CXCR4 alone [X4]) emerge at late-stage infection in half of HIV-1-infected individuals and are associated with disease progression. Although X4 variants also appear during acute-phase infection in some cases, these variants apparently fall to undetectable levels thereafter. In this study, replication-competent X4 variants were isolated from plasma of drug treatment-naive individuals infected with HIV-1 strain CRF01_AE, which dominantly carries viral RNA (vRNA) of R5 variants. Next-generation sequencing (NGS) confirmed that sequences of X4 variants were indeed present in plasma vRNA from these individuals as a minor population. On the other hand, in one individual with a mixed infection in which X4 variants were dominant, only R5 replication-competent variants were isolated from plasma. These results indicate the existence of replication-competent variants with different coreceptor usage as minor populations.IMPORTANCE The coreceptor switch of HIV-1 from R5 to CXCR4-using variants (R5X4 or X4) has been observed in about half of HIV-1-infected individuals at late-stage infection with loss of CD4 cell count and disease progression. However, the mechanisms that underlie the emergence of CXCR4-using variants at this stage are unclear. In the present study, CXCR4-using X4 variants were isolated from plasma samples of HIV-1-infected individuals that dominantly carried vRNA of R5 variants. The sequences of the X4 variants were detected as a minor population using next-generation sequencing. Taken together, CXCR4-using variants at late-stage infection are likely to emerge when replication-competent CXCR4-using variants are maintained as a minor population during the course of infection. The present study may support the hypothesis that R5-to-X4 switching is mediated by the expansion of preexisting X4 variants in some cases.

Prediction of Contact Residues in Anti-HIV Neutralizing Antibody by Deep Learning.

The monoclonal antibody 1C10 targets the V3 loop of HIV-1 and neutralizes a broad range of clade B viruses. However, the mode of interaction between 1C10 and the V3 loop remains unclear because crystallization of 1C10 and the V3 peptide was unsuccessful due to the flexible regions present in both 1C10 and the V3 peptide. In this study, we predicted the 1C10 amino acid residues that make contact with the V3 loop using a deep learning (DL)-based method. Inputs from ROSIE for docking simulation and FastContact, Naccess, and PDBtools, to approximate interactions were processed by Chainer for DL, and outputs were obtained as probabilities of contact residues. Using this DL algorithm, D95, D97, P100a, and D100b of CDRH3; D53, and D56 of CDRH2; and D61 of FR3 were highly ranked as contact residues of 1C10. Substitution of these residues with alanine significantly decreased the affinity of 1C10 to the V3 peptide. Moreover, the higher the rank of the residue, the more the binding activity diminished. This study demonstrates that the prediction of contact residues using a DL-based approach is a precise and useful tool for the analysis of antibody-antigen interactions.

Synergistic inhibition of cell-to-cell HIV-1 infection by combinations of single chain variable fragments and fusion inhibitors.

Cell-to-cell spread of HIV permits ongoing viral replication in the presence of antiretroviral therapy and is suggested to be a major contributor to sexual transmission by mucosal routes. Fusion inhibitors that prevent viral entry have been developed, but their clinical applications have been limited by weak antiviral activity, short half-life, and the low genetic barrier to development of resistance. We examined the inhibitory activities of a series of single-chain variable fragments (scFvs) targeting the V3 and CD4i epitopes against both cell-free and cell-to-cell HIV infection. We found that all anti-V3 scFvs, including two newly constructed scFvs, showed broad neutralization activity against a panel of subtype B viruses compared with the corresponding IgGs. All scFvs neutralized cell-free infection by HIV-1JR-FL WT and fusion inhibitor-resistant mutants. In addition, all anti-V3 scFvs and some CD4i scFvs significantly inhibited cell fusion, while their IgG counterparts did not. Furthermore, scFvs-fusion inhibitors combinations, such as C34 and SC34, showed synergistic inhibition of cell fusion by both HIV-1JR-FL WT and fusion inhibitor-resistant mutants. The most prominent combinational effect was observed for 916B2 CD4i scFv with SC34. The delayed fusion kinetics of fusion inhibitor-resistant mutants partly explain their synergistic inhibition by such combinations. Our data demonstrate the advantages of using scFvs over their parent IgGs for inhibiting both cell-free and cell-to-cell infection. High synergistic inhibition of cell fusion by using scFvs-fusion inhibitors combinations suggests the possibility of intensification therapy adding this combination to current anti-HIV treatment regimens.

The role of conventional antibodies targeting the CD4 binding site and CD4-induced epitopes in the control of HIV-1 CRF01_AE viruses.

HIV-1 CRF01_AE viruses are highly prevalent in Southeast Asia. However, vulnerability sites in Env of CRF01_AE viruses have not been investigated sufficiently. We examined the sensitivity of CRF01_AE viruses from Japan and Vietnam, together with subtype B viruses from Japan, to neutralization and Fc-mediated signaling. Neutralization coverage of broadly neutralizing antibodies (bnAbs), 2G12 and b12, was significantly low against CRF01_AE viruses, compared with subtype B viruses. In contrast, the conventional antibody targeting the CD4 binding site (CD4bs), 49G2, showed better neutralization and Fc-mediated signaling activities against CRF01_AE viruses than subtype B viruses. Fc-mediated signaling activity of anti-CD4 induced (CD4i) antibody, 4E9C, was also detected against CRF01_AE viruses more than subtype B viruses. These results suggest that conventional antibodies against CD4bs and CD4i may play an important role in the control of CRF01_AE viruses.

Total HIV-1 DNA Dynamics and Influencing Factors in Long-Term ART-Treated Japanese Adults: A Retrospective Longitudinal Analysis.

BACKGROUND: Understanding HIV persistence in treated patients is an important milestone toward drug-free control. We aimed at analyzing total HIV DNA dynamics and influencing factors in Japanese patients who received more than a decade of suppressive antiretroviral treatment (ART). METHODS: A retrospective study including clinical records and 840 peripheral blood mononuclear cells samples (mean 14 samples/patient) for 59 patients (92% male) was performed. Subjects were divided into 2 groups: with and without hematological comorbidity (mainly hemophilia) plus hepatitis C virus coinfection. Total HIV DNA was measured in peripheral blood mononuclear cells by quantitative polymerase chain reaction. The dynamics, regression over time, and influence of antiretrovirals by group were estimated using a novel regression model (R software v 3.2.3). RESULTS: Total HIV DNA decreased on ART initiation, and subsequently, its dynamics varied between groups with previously undescribed fluctuations. If calculated by on-treatment, the mean total HIV DNA levels were similar. The comorbidity group had unstable levels showing different regression over time (P = 0.088/0.094 in year 1/after year 8 of ART) and significantly different treatment responses as shown by antiretroviral group switching estimates. Furthermore, curing hepatitis C virus in hemophiliacs did not significantly alter total HIV DNA levels or regression. CONCLUSIONS: Our data identified some effects of the long-term treatment on total HIV DNA levels and highlighted the partial influence of comorbidities and coinfections. Total HIV DNA monitoring contributed to therapy response estimates and HIV reservoir quantification. The results suggest that HIV DNA monitoring during ART might be useful as a persistence marker in both HIV-monoinfected patients and those with comorbidities and coinfections.

Unique binding modes for the broad neutralizing activity of single-chain variable fragments (scFv) targeting CD4-induced epitopes.

BACKGROUND: The CD4-induced (CD4i) epitopes in gp120 includes the co-receptor binding site, which are formed and exposed after interaction with CD4. Monoclonal antibodies (mAbs) to the CD4i epitopes exhibit limited neutralizing activity because of restricted access to their epitopes. However, small fragment counterparts such as single-chain variable fragments (scFvs) have been reported to neutralize a broad range of viruses compared with the full-size IgG molecule. To identify the CD4i epitope site responsible for this broad neutralization we constructed three scFvs of anti-CD4i mAbs from a human immunodeficiency virus type 1 (HIV-1)-infected elite controller, and investigated the neutralization coverage and precise binding site in the CD4i epitopes. RESULTS: We constructed scFvs from the anti-CD4i mAbs, 916B2, 4E9C, and 25C4b and tested their neutralization activity against a panel of 66 viruses of multi-subtype. Coverage of neutralization by the scFvs against this panel of pseudoviruses was 89% (59/66) for 4E9C, 95% (63/66) for 25C4b and 100% (66/66) for 916B2. Analysis using a series of envelope glycoprotein mutants revealed that individual anti-CD4i mAbs showed various dependencies on the hairpin 1 (H1) and V3 base. The binding profiles of 25C4b were similar to those of 17b, and 25C4b bound the region spanning multiple domains of H1 and hairpin 2 (H2) of the bridging sheet and V3 base. For 4E9C, the V3-base dependent binding was apparent from no binding to mutants containing the ΔV3 truncation. In contrast, binding of 916B2 was dependent on the H1 region, which is composed of ß2 and ß3 strands, because mutants containing the H1 truncation did not show any reactivity to 916B2. Although the H1 region structure is affected by CD4 engagement, the results indicate the unique nature of the 916B2 epitope, which may be structurally conserved before and after conformational changes of gp120. CONCLUSIONS: Identification of a unique structure of the H1 region that can be targeted by 916B2 may have an important implication in the development of small molecules to inhibit infection by a broad range of HIV-1 for the purpose of HIV treatment and prevention.

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.

Cross-Neutralization Activity of Single-Chain Variable Fragment (scFv) Derived from Anti-V3 Monoclonal Antibodies Mediated by Post-Attachment Binding.

The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed single-chain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5γ, 5G2, and 16G6. Enhanced neutralization by 0.5γ and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5γ scFv reached up to 90% of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5γ scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an inter-subunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after attachment was 60 to 120 min. These results indicate that the scFvs efficiently access the V3 loop and subsequently neutralize HIV-1, even after virus attachment to the target cells. Based on its broad and potent neutralizing activity, further development of anti-V3 scFv for therapeutic and preventive strategies is warranted.