Exceptionally Potent and Broadly Cross-Reactive,Bispecific Multivalent HIV-1 Inhibitors Based on Single Human CD4 and Antibody Domains

Soluble forms of the human immunodeficiency virus type 1 (HIV-1) primary receptor CD4 (soluble CD4 [sCD4]) have been extensively characterized for a quarter of a century as promising HIV-1 inhibitors, but they have not been clinically successful. By combining a protein cavity-filling strategy and the power of library technology, we identified an engineered cavity-altered single-domain sCD4 (mD1.22) with a unique combination of excellent properties, including broad and potent neutralizing activity, high specificity, stability, solubility, and affinity for the HIV-1 envelope glycoprotein gp120, and small molecular size. To further improve its neutralizing potency and breadth, we generated bispecific multivalent fusion proteins of mD1.22 with another potent HIV-1 inhibitor, an antibody domain (m36.4) that targets the coreceptor-binding site on gp120. The fusion proteins neutralized all HIV-1 isolates tested, with potencies about 10-, 50-, and 200-fold higher than those of the broadly neutralizing antibody VRC01, the U.S. FDA-approved peptide inhibitor T20, and the clinically tested sCD4-Fc fusion protein CD4-Ig, respectively. In addition, they exhibited higher stability and specificity and a lower aggregation propensity than CD4-Ig. Therefore, mD1.22 and related fusion proteins could be useful for HIV-1 prevention and therapy, including eradication of the virus.     

Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics.     

Species-Specific Activity of HIV-1 Vpu and Positive Selection of Tetherin Transmembrane Domain Variants

Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins.     

Immunization of Rabbits with Highly Purified,Soluble, Trimeric Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Induces a Vigorous B Cell Response and Broadly Cross-Reactive Neutralization

Previously we described induction of cross-reactive HIV-1 neutralizing antibody responses in rabbits using a soluble HIV-1 gp140 envelope glycoprotein (Env) in an adjuvant containing monophosphoryl lipid A (MPL) and QS21 (AS02_A). Here, we compared different forms of the same HIV-1 strain R2 Env for antigenic and biophysical characteristics, and in rabbits characterized the extent of B cell induction for specific antibody expression and secretion and neutralizing responses. The forms of this Env that were produced in and purified from stably transformed 293T cells included a primarily dimeric gp140, a trimeric gp140 appended to a GCN4 trimerization domain (gp140-GCN4), gp140-GCN4 with a 15 amino acid flexible linker between the gp120 and gp41 ectodomain (gp140-GCN4-L), also trimeric, and a gp140 with the flexible linker purified from cell culture supernatants as either dimer (gp140-L(D)) or monomer (gp140-L(M)). Multimeric states of the Env proteins were assessed by native gel electrophoresis and analytical ultracentrifugation. The different forms of gp140 bound broadly cross-reactive neutralizing (BCN) human monoclonal antibodies (mAbs) similarly in ELISA and immunoprecipitation assays. All Envs bound CD4i mAbs in the presence and absence of sCD4, as reported for the R2 Env. Weak neutralization of some strains of HIV-1 was seen after two additional doses in AS02_A. Rabbits that were given a seventh dose of gp140-GCN4-L developed BCN responses that were weak to moderate, similar to our previous report. The specificity of these responses did not appear similar to that of any of the known BCN human mAbs. Induction of spleen B cell and plasma cells producing immunoglobulins that bound trimeric gp140-GCN4-L was vigorous, based on ELISpot and flow cytometry analyses. The results demonstrate that highly purified gp140-GCN4-L trimer in adjuvant elicits BCN responses in rabbits accompanied by vigorous B cell induction.     

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA: Diagnostic Accuracy,Viral Evolution and Compartmentalization

Background

Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain.

Methods & Results

We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized.

Conclusions

The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.     

B Cell Responses to HIV Antigen Are a Potent Correlate of Viremia in HIV-1 Infection and Improve with PD-1 Blockade

Infection with Human Immunodeficiency Virus Type 1 (HIV-1) induces defects of both cellular and humoral immune responses. Impaired CD4+ T cell help and B cell dysfunction may partially explain the low frequency of broadly neutralizing antibodies in HIV-infected individuals. To understand the extent of B cell dysfunction during HIV infection, we assessed the level of B cell activation at baseline and after stimulation with a variety of antigens. Increased levels of viremia were associated with higher baseline expression of the activation marker CD86 on B cells and with decreased ability of B cells to increase expression of CD86 after in vitro stimulation with inactivated HIV-1. In a series of cell isolation experiments B cell responses to antigen were enhanced in the presence of autologous CD4+ T cells. HIV infected individuals had a higher frequency of PD-1 expression on B cells compared to HIV- subjects and PD-1 blockade improved B cell responsiveness to HIV antigen, suggesting that inhibitory molecule expression during HIV-1 infection may contribute to some of the observed B cell defects. Our findings demonstrate that during chronic HIV infection, B cells are activated and lose full capacity to respond to antigen, but suppression of inhibitory pressures as well as a robust CD4+ T cell response may help preserve B cell function.     

Levels of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T-Lymphocyte Effector and Memory Responses Decline after Suppression of Viremia with Highly Active Antiretroviral Therapy

Therapeutic suppression of human immunodeficiency virus type 1 (HIV-1) replication may help elucidate interactions between the host cellular immune responses and HIV-1 infection. We performed a detailed longitudinal evaluation of two subjects before and after the start of highly active antiretroviral therapy (HAART). Both subjects had evidence of in vivo-activated and memory cytotoxic T-lymphocyte precursor (CTLp) activity against multiple HIV-1 gene products. After the start of therapy, both subjects had declines in the levels of in vivo-activated HIV-1-specific CTLs and had immediate increases in circulating HIV-1-specific CTL memory cells. With continued therapy, and continued suppression of viral load, levels of memory CTLps declined. HLA A*0201 peptide tetramer staining demonstrated that declining levels of in vivo-activated CTL activity were associated with a decrease in the expression of the CD38(+) activation marker. Transient increases in viral load during continued therapy were associated with increases in the levels of virus-specific CTLps in both individuals. The results were confirmed by measuring CTL responses to discrete optimal epitopes. These studies illustrate the dynamic equilibrium between the host immune response and levels of viral antigen burden and suggest that efforts to augment HIV-1-specific immune responses in subjects on HAART may decrease the incidence of virologic relapse.     

Effectiveness of a Treatment Switch to Nevirapine plus Tenofovir and Emtricitabine (or Lamivudine) in Adults with HIV-1 Suppressed Viremia

Background

Switching subjects with persistently undetectable HIV-1 viremia under antiretroviral treatment (ART) to once-daily tenofovir/emtricitabine (or lamivudine) + nevirapine is a cost-effective and well-tolerated strategy. However, the effectiveness of this approach has not been established.

Methods

We performed a retrospective study evaluating the rates of treatment failure, virological failure (VF), and variables associated, in all subjects initiating this switch combination in our clinic since 2001. Analyses were performed by a modified intention to treat, where switch due to toxicity equalled failure. The main endpoint was plasma HIV-RNA < 50 copies/mL.

Results

341 patients were treated for a median of 176 (57; 308) weeks. At week 48, 306 (89.7%) subjects had HIV-1 RNA <50 copies/mL, 10 (2.9%) experienced VF, and 25 (7.4%) discontinued the treatment due to toxicity. During the whole follow-up 23 (6.7%) individuals (17 on lamivudine, 6 on emtricitabine; p = 0.034) developed VF and treatment modification due to toxicity occurred in 36 (10.7%). Factors independently associated with VF in a multivariate analysis were: intravenous drug use (HR 1.51; 95%CI 1.12, 2.04), time with undetectable viral load before the switch (HR 0.98; 0.97, 0.99), number of prior NRTIs (HR 1.49; 1.15, 1.93) or NNRTIs (HR 3.22; 1.64, 6.25), and previous NVP (HR 1.54; 1.10, 2.17) or efavirenz (HR 5.76; 1.11, 29.87) unscheduled interruptions. VF was associated with emergence of usual nevirapine mutations (Y181C/I/D, K103N and V106A/I), M184V (n = 16; 12 with lamivudine vs. 4 with emtricitabine, p = 0.04), and K65R (n = 7).

Conclusions

The rates of treatment failure at 48 weeks, or long-term toxicity or VF with this switch regimen are low and no unexpected mutations or patterns of mutations were selected in subjects with treatment failure.     

丝瓜籽中一种具有翻译抑制活性和胰蛋白酶抑制剂活性的多肽——Luffin P1的纯化和性质

通过硫酸铵分级沉淀、CM 5 2阳离子交换层析、蓝胶亲和层析和FPLCMonoS阳离子交换层析 ,从丝瓜籽抽提液中分离到一种多肽luffinP1。经MALDI TOFMS测得其分子量为 5 2 2 6 .5。氨基酸序列测定及同源性分析发现 ,luffinP1的N端 11个氨基酸序列与丝瓜籽中的一种 6 .5K富含Arg Glu的多肽AGRP的部分序列相同 ,并与南瓜籽中一种胰蛋白酶抑制剂C2肽具有很高的同源性。体外分析表明 ,luffinP1同时具有两种生物活性 :(1)对兔网织红细胞裂解液系统蛋白质生物合成有较强的抑制作用 ,IC50 为 0 .6nmol L ;(2 )具有明显的胰蛋白酶抑制活性 ,IC50 为 2 2 μmol L。     

Evolution of viruses and their impact on human life: HIV-1 subgroup M

The history of mankind over many millennia has been marred by many epidemics caused by viruses which infect the human respiratory system and alimentary tract. The current HIV-1/AIDS pandemic, however, is caused by one virus mutant, HIV-1M, which has evolved to infect humans through the genitals. The virus is able to use the innate system cells of the infected individual to inactivate the adaptive immune system, causing AIDS. The mechanisms used by HIV-1M to inhibit the immune system are presented. Understanding the viral mechanisms is leading to novel antiviral treatments and an approach to an HIV-1 vaccine.     

Epitope Mapping of M36, a Human Antibody Domain with Potent and Broad HIV-1 Inhibitory Activity

M36 is the first member of a novel class of potent HIV-1 entry inhibitors based on human engineered antibody domains (eAds). It exhibits broad inhibitory activity suggesting that its CD4-induced epitope is highly conserved. Here, we describe fine mapping of its epitope by using several approaches. First, a panel of mimotopes was affinity-selected from a random peptide library and potential m36-binding residues were computationally predicted. Second, homology modeling of m36 and molecular docking of m36 onto gp120 revealed potentially important residues in gp120-m36 interactions. Third, the predicted contact residues were verified by site-directed mutagenesis. Taken together, m36 epitope comprising three discontinuous sites including six key gp120 residues (Site C1: Thr123 and Pro124; Site C3: Glu370 and Ile371; Site C4: Met426 and Trp427) were identified. In the 3D structure of gp120, the sites C1 and C4 are located in the bridging sheet and the site C3 is within the β15-α3 excursion, which play essential roles for the receptor- and coreceptor-binding and are major targets of neutralizing antibodies. Based on these results we propose a precise localization of the m36 epitope and suggest a mechanism of its broad inhibitory activity which could help in the development of novel HIV-1 therapeutics based on eAds.     

Loss of the Protease Dimerization Inhibition Activity of Tipranavir (TPV) and Its Association with the Acquisition of Resistance to TPV by HIV-1

Tipranavir (TPV), a protease inhibitor (PI) inhibiting the enzymatic activity and dimerization of HIV-1 protease, exerts potent activity against multi-PI-resistant HIV-1 isolates. When a mixture of 11 multi-PI-resistant (but TPV-sensitive) clinical isolates (HIV_11MIX), which included HIV_B and HIV_C, was selected against TPV, HIV_11MIX rapidly (by 10 passages [HIV_11MIX^P10]) acquired high-level TPV resistance and replicated at high concentrations of TPV. HIV_11MIX^P10 contained various amino acid substitutions, including I54V and V82T. The intermolecular FRET-based HIV-1 expression assay revealed that TPV''s dimerization inhibition activity against cloned HIV_B (cHIV_B) was substantially compromised. The introduction of I54V/V82T into wild-type cHIV_NL4-3 (cHIV_{NL4-3^I54V/V82T}) did not block TPV''s dimerization inhibition or confer TPV resistance. However, the introduction of I54V/V82T into cHIV_B (cHIV_B^I54V/V82T) compromised TPV''s dimerization inhibition and cHIV_B^I54V/V82T proved to be significantly TPV resistant. L24M was responsible for TPV resistance with the cHIV_C genetic background. The introduction of L24M into cHIV_NL4-3 (cHIV_NL4-3^L24M) interfered with TPV''s dimerization inhibition, while L24M increased HIV-1''s susceptibility to TPV with the HIV_NL4-3 genetic background. When selected with TPV, cHIV_{NL4-3^I54V/V82T} most readily developed TPV resistance and acquired E34D, which compromised TPV''s dimerization inhibition with the HIV_NL4-3 genetic background. The present data demonstrate that certain amino acid substitutions compromise TPV''s dimerization inhibition and confer TPV resistance, although the loss of TPV''s dimerization inhibition is not always associated with significantly increased TPV resistance. The findings that TPV''s dimerization inhibition is compromised with one or two amino acid substitutions may explain at least in part why the genetic barrier of TPV against HIV-1''s development of TPV resistance is relatively low compared to that of darunavir.     

Characterization of LEDGF/p75 Genetic Variants and Association with HIV-1 Disease Progression

Background

As Lens epithelium-derived growth factor (LEDGF/p75) is an important co-factor involved in HIV-1 integration, the LEDGF/p75-IN interaction is a promising target for the new class of allosteric HIV integrase inhibitors (LEDGINs). Few data are available on the genetic variability of LEDGF/p75 and the influence on HIV disease in vivo. This study evaluated the relation between LEDGF/p75 genetic variation, mRNA expression and HIV-1 disease progression in order to guide future clinical use of LEDGINs.

Methods

Samples were derived from a therapy-naïve cohort at Ghent University Hospital and a Spanish long-term-non-progressor cohort. High-resolution melting curve analysis and Sanger sequencing were used to identify all single nucleotide polymorphisms (SNPs) in the coding region, flanking intronic regions and full 3′UTR of LEDGF/p75. In addition, two intronic tagSNPs were screened based on previous indication of influencing HIV disease. LEDGF/p75 mRNA was quantified in patient peripheral blood mononuclear cells (PBMC) using RT-qPCR.

Results

325 samples were investigated from patients of Caucasian (n = 291) and African (n = 34) origin, including Elite (n = 49) and Viremic controllers (n = 62). 21 SNPs were identified, comprising five in the coding region and 16 in the non-coding regions and 3′UTR. The variants in the coding region were infrequent and had no major impact on protein structure according to SIFT and PolyPhen score. One intronic SNP (rs2737828) was significantly under-represented in Caucasian patients (P<0.0001) compared to healthy controls (HapMap). Two SNPs showed a non-significant trend towards association with slower disease progression but not with LEDGF/p75 expression. The observed variation in LEDGF/p75 expression was not correlated with disease progression.

Conclusions

LEDGF/p75 is a highly conserved protein. Two non-coding polymorphisms were identified indicating a correlation with disease outcome, but further research is needed to clarify phenotypic impact. The conserved coding region and the observed variation in LEDGF/p75 expression are important characteristics for clinical use of LEDGINs.     

Association of TRIM22 with the type 1 interferon response and viral control during primary HIV-1 infection

Type 1 interferons (IFNs) induce the expression of the tripartite interaction motif (TRIM) family of E3 ligases, but the contribution of these antiviral factors to HIV pathogenesis is not completely understood. We hypothesized that the increased expression of select type 1 IFN and TRIM isoforms is associated with a significantly lower likelihood of HIV-1 acquisition and viral control during primary HIV-1 infection. We measured IFN-α, IFN-β, myxovirus resistance protein A (MxA), human TRIM5α (huTRIM5α), and TRIM22 mRNA levels in peripheral blood mononuclear cells (PBMCs) of high-risk, HIV-1-uninfected participants and HIV-1-positive study participants. Samples were available for 32 uninfected subjects and 28 infected persons, all within 1 year of infection. HIV-1-positive participants had higher levels of IFN-β (P = 0.0005), MxA (P = 0.007), and TRIM22 (P = 0.01) and lower levels of huTRIM5α (P < 0.001) than did HIV-1-negative participants. TRIM22 but not huTRIM5α correlated positively with type 1 IFN (IFN-α, IFN-β, and MxA) (all P < 0.0001). In a multivariate model, increased MxA expression showed a significant positive association with viral load (P = 0.0418). Furthermore, TRIM22 but not huTRIM5α, IFN-α, IFN-β, or MxA showed a negative correlation with plasma viral load (P = 0.0307) and a positive correlation with CD4(+) T-cell counts (P = 0.0281). In vitro studies revealed that HIV infection induced TRIM22 expression in PBMCs obtained from HIV-negative donors. Stable TRIM22 knockdown resulted in increased HIV-1 particle release and replication in Jurkat reporter cells. Collectively, these data suggest concordance between type 1 IFN and TRIM22 but not huTRIM5α expression in PBMCs and that TRIM22 likely acts as an antiviral effector in vivo.     

Dengue in Travelers: Kinetics of Viremia and NS1 Antigenemia and Their Associations with Clinical Parameters

Despite the increasing numbers of travel-acquired dengue, few studies have assessed virologic markers of the disease in non-endemic populations. We examined the kinetics of diagnostic markers and their associations with clinical parameters in 93 patients with travel-acquired dengue fever. Kinetics analyses suggested a longer average duration for viremia (9 days, CI95%: 8–10) and non-structural protein 1 (NS1) antigenemia (15 days, CI95%: 12–20) than reported in endemic populations. While none of the tests sufficed alone, the best diagnostic coverage was achieved by combining antibody detection with RNA or NS1 testing. Studied by regression models, early relative levels of viremia and NS1 antigenemia proved to be significantly associated with several clinical parameters: high viremia predicted greater likelihood and increased length of hospitalization, the degree of NS1 antigenemia correlated positively with hematocrit and liver transaminases, and both viremia and NS1 antigenemia levels negatively with platelet counts in follow-up. Levels of viremia and NS1 antigenemia may serve as predictors of the clinical manifestations in travel-acquired dengue.     

Sequences in Glycoprotein gp41, the CD4 Binding Site,and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals.     

A Variable Region 3 (V3) Mutation Determines a Global Neutralization Phenotype and CD4-Independent Infectivity of a Human Immunodeficiency Virus Type 1 Envelope Associated with a Broadly Cross-Reactive, Primary Virus-Neutralizing Antibody Response

The human serum human immunodeficiency virus type 1 (HIV-1)-neutralizing serum 2 (HNS2) neutralizes many primary isolates of different clades of HIV-1, and virus expressing envelope from the same donor, clone R2, is neutralized cross-reactively by HIV-immune human sera. The basis for this cross-reactivity was investigated. It was found that a rare mutation in the proximal limb of variable region 3 (V3), 313-4 PM, caused virus pseudotyped with the R2 envelope to be highly sensitive to neutralization by monoclonal antibodies (MAbs) directed against conformation-sensitive epitopes at the tip of the V3 loop, such as 19b, and moderately sensitive to MAbs against CD4 binding site (CD4bs) and CD4-induced (CD4i) epitopes, soluble CD4 (sCD4), and HNS2. In addition, introduction of this sequence by mutagenesis caused enhanced sensitivity to neutralization by 19b, anti-CD4i MAb, and HNS2 in three other primary HIV-1 envelopes and by anti-CD4bs MAb and sCD4 in one of the three. The 313-4 PM sequence also conferred increased infectivity for CD4(+) CCR5(+) cells and the ability to infect CCR5(+) cells upon all of these four and two of these four HIV-1 envelopes, respectively. Neutralization of R2 by HNS2 was substantially inhibited by the cyclized R2 V3 35-mer synthetic peptide. Similarly, the peptide also had some lesser efficacy in blocking neutralization of R2 by other sera or of neutralization of other primary viruses by HNS2. Together, these results indicate that the unusual V3 mutation in the R2 clone accounts for its uncommon neutralization sensitivity phenotype and its capacity to mediate CD4-independent infection, both of which could relate to immunogenicity and the neutralizing activity of HNS2. This is also the first primary HIV-1 isolate envelope glycoprotein found to be competent for CD4-independent infection.