Autologous and heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals.

In the course of human immunodeficiency virus type 1 (HIV-1) infection, patients develop a strong and persistent immune response characterized by the production of HIV-specific antibodies. The aim of our study was to analyze the appearance of autologous and heterologous neutralizing antibodies in the sera of HIV-infected individuals. For this purpose, primary strains have been isolated from 18 HIV-1-infected subjects prior to seroconversion (in one case) or within 1 to 8 months after seroconversion. Sera, collected at the same time as the virus was isolated and at various times after isolation, have been analyzed for their ability to neutralize the autologous primary strains isolated early after infection, heterologous primary isolates, and cell-line adapted strains. Our neutralization assay, which combines serial dilutions of virus and serial dilutions of sera, is based on the determination of the serum dilution at which a fixed reduction in virus titer (90%) occurs. We have shown that (i) we could not detect autologous neutralizing antibodies in sera collected at the same time as we isolated viruses; (ii) we detected neutralizing antibodies against the autologous strains about 1 year after seroconversion, occasionally after 8 months, but sera were not always available to exclude the presence of neutralizing antibodies at earlier times; (iii) after 1 year, the neutralization response was highly specific to virus present during the early phase of HIV infection; and (iv) heterologous neutralization of primary isolates was detected later (after about 2 years). These results reveal the enormous diversity of neutralization determinants on primary isolates as well as a temporal evolution of the humoral response generating cross-reactive neutralizing antibodies.     

Neutralizing antibody responses against autologous and heterologous viruses in acute versus chronic human immunodeficiency virus (HIV) infection: evidence for a constraint on the ability of HIV to completely evade neutralizing antibody responses

Acute human immunodeficiency virus (HIV) infection is associated with the rapid development of neutralization escape mutations. The degree to which viral evolution persists in chronic infection has not been well characterized, nor is it clear if all patients develop high-level neutralization antibody escape. We therefore measured neutralizing antibody responses against autologous and heterologous viruses in a cohort of acutely and chronically infected subjects (n = 65). Neutralizing antibody responses against both autologous virus and heterologous viruses were lower among individuals with acute infection than among those with chronic infection. Among chronically infected individuals, there was a negative correlation between the level of neutralizing antibodies against autologous virus and the level of viremia. In contrast, there was a positive correlation between the level of neutralizing antibodies against a panel of heterologous viruses and the level of viremia. Viral evolution, as defined by the presence of higher neutralizing titers directed against earlier viruses than against contemporaneous viruses, was evident for subjects with recent infection but absent for those with chronic infection. In summary, neutralizing antibody responses against contemporaneous autologous viruses are absent in early HIV infection but can be detected at low levels in chronic infection, particularly among those controlling HIV in the absence of therapy. HIV replication either directly or indirectly drives the production of increasing levels of antibodies that cross-neutralize heterologous primary isolates. Collectively, these observations indicate that although HIV continuously drives the production of neutralizing antibodies, there may be limits to the capacity of the virus to evolve continuously in response to these antibodies. These observations also suggest that the neutralizing antibody response may contribute to the long-term control of HIV in some patients while protecting against HIV superinfection in most patients.     

The c3-v4 region is a major target of autologous neutralizing antibodies in human immunodeficiency virus type 1 subtype C infection

The early autologous neutralizing antibody response in human immunodeficiency virus type 1 (HIV-1) subtype C infections is often characterized by high titers, but the response is type specific with little to no cross-neutralizing activity. The specificities of these early neutralizing antibodies are not known; however, the type specificity suggests that they may target the variable regions of the envelope. Here, we show that cross-reactive anti-V3 antibodies developed within 3 to 12 weeks in six individuals but did not mediate autologous neutralization. Using a series of chimeric viruses, we found that antibodies directed at the V1V2, V4, and V5 regions contributed to autologous neutralization in some individuals, with V1V2 playing a more substantial role. However, these antibodies did not account for the total neutralizing capacity of these sera against the early autologous virus. Antibodies directed against the C3-V4 region were involved in autologous neutralization in all four sera studied. In two sera, transfer of the C3-V4 region rendered the chimera as sensitive to antibody neutralization as the parental virus. Although the C3 region, which contains the highly variable α2-helix was not a direct target in most cases, it contributed to the formation of neutralization epitopes as substitution of this region resulted in neutralization resistance. These data suggest that the C3 and V4 regions combine to form important structural motifs and that epitopes in this region are major targets of the early autologous neutralizing response in HIV-1 subtype C infection.     

Autologous neutralizing humoral immunity and evolution of the viral envelope in the course of subtype B human immunodeficiency virus type 1 infection

Most human immunodeficiency virus type 1 (HIV-1)-infected individuals develop an HIV-specific neutralizing antibody (NAb) response that selects for escape variants of the virus. Here, we studied autologous NAb responses in five typical CCR5-using progressors in relation to viral NAb escape and molecular changes in the viral envelope (Env) in the period from seroconversion until after AIDS diagnosis. In sera from three patients, high-titer neutralizing activity was observed against the earliest autologous virus variants, followed by declining humoral immune responses against subsequent viral escape variants. Autologous neutralizing activity was undetectable in sera from two patients. Patients with high-titer neutralizing activity in serum showed the strongest positive selection pressure on Env early in infection. In the initial phase of infection, gp160 length and the number of potential N-linked glycosylation sites (PNGS) increased in viruses from all patients. Over the course of infection, positive selection pressure declined as the NAb response subsided, coinciding with reversions of changes in gp160 length and the number of PNGS. A number of identical amino acid changes were observed over the course of infection in the viral quasispecies of different patients. Our results indicate that although neutralizing autologous humoral immunity may have a limited effect on the disease course, it is an important selection pressure in virus evolution early in infection, while declining HIV-specific humoral immunity in later stages may coincide with reversion of NAb-driven changes in Env.     

Structure and design of broadly-neutralizing antibodies against HIV

Since the discovery more than 30 years ago of human immunodeficiency virus (HIV) as the causative agent of the deadly disease, acquired immune deficiency disease (AIDS), there have been no efficient vaccines against the virus. For the infection of the virus, the HIV surface glycoprotein gp120 first recognizes the CD4 receptor on the target helper T-cell, which initiates HIV fusion with the target cell and, if unchecked, leads to destruction of the patient's immune system. Despite the difficulty of developing appropriate immune responses in HIV-infected individuals, patient sera often contain antibodies that have broad neutralization activity, indicating the possibility of immunological treatment and prevention. Recently, through extensive structural studies of neutralizing antibodies of HIV in complex with gp120, the critical mechanisms of broad neutralization against HIV have been elucidated. Based on these discoveries, the structure-aided designs of antibodies and novel scaffolds were performed to create extremely potent neutralizing antibodies against HIV. These new discoveries and advances shed light on the road to development of efficient immunological therapies against AIDS.     

＂Unconventional＂ Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However,only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes,such as high sequence diversity,heavy glycosylation,and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohe-magglutinin (PHA)-stimulated human PBMCs as target cells. Thus,in essence the assay evaluates HIV-1 replication in CD4 T cells. Recently,several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera,although they do not have neutralizing activity when tested by the "conventional" neutralization assay,do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications,through opsonization of virus particles into macrophages and immature dendritic cells (iDCs),or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.     

"Unconventional" Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.     

Viremia control despite escape from a rapid and potent autologous neutralizing antibody response after therapy cessation in an HIV-1-infected individual

The neutralizing Ab response after primary HIV-1 infection is delayed relative to the virus-specific CD8(+) T cell response and the initial decline in plasma viremia. Because nearly all HIV-1 infections result in AIDS, it would be instructive to study cases where neutralizing Ab production commenced sooner. This was done in subject AC10, an individual treated during early infection and in whom a rapid autologous neutralizing Ab response was detected after therapy cessation as rebound viremia declined and remained below 1000 RNA copies/ml of blood for over 2.5 years. This subject's Abs were capable of reducing the infectivity of his rebound virus by >4 logs in vitro at a time when rebound viremia was down-regulated and virus-specific CD8(+) T cells were minimal, suggesting that neutralizing Abs played an important role in the early control of viremia. The rebound virus did not exhibit an unusual phenotype that might explain its high sensitivity to neutralization by autologous sera. Neutralization escape occurred within 75 days and was proceeded by neutralizing Ab production to the escape variant and subsequent escape. Notably, escape was not associated with a significant rise in plasma viremia, perhaps due to increasing CD8(+) T cell responses. Sequence analysis of gp160 revealed a growing number of mutations over time, suggesting ongoing viral evolution in the face of potent antiviral immune responses. We postulate that an early effective neutralizing Ab response can provide long-term clinical benefits despite neutralization escape.     

A Nonparametric Procedure for Defining a New Humoral Immunologic Profile in a Pilot Study on HIV Infected Patients

This work aims at identifying a set of humoral immunologic parameters that improve prediction of the activation process in HIV patients. Starting from the well-known impact of humoral immunity in HIV infection, there is still a lack of knowledge in defining the role of the modulation of functional activity and titers of serum antibodies from early stage of infection to the development of AIDS. We propose an integrated approach that combines humoral and clinical parameters in defining the host immunity, implementing algorithms associated with virus control. A number of humoral parameters were simultaneously evaluated in a whole range of serum samples from HIV-positive patients. This issue has been afforded accounting for estimation problems typically related to “feasibility” studies where small sample size in each group and large number of parameters are jointly estimated. We used nonparametric statistical procedures to identify biomarkers in our study which included 42 subjects stratified on five different stages of HIV infection, i.e., Elite Controllers (EC), Long Term Non Progressors (LTNP), HAART, AIDS and Acute Infection (AI). The main goal of the paper is to illustrate a novel profiling method for helping to design a further confirmatory study. A set of seventeen different HIV-specific blood humoral factors were analyzed in all subjects, i.e. IgG and IgA to gp120IIIB, to gp120Bal, to whole gp41, to P1 and T20 gp41 epitopes of the MPER-HR2 region, to QARILAV gp41 epitope of the HR1 region and to CCR5; neutralization activity against five different virus strains and ADCC were also evaluated. Patients were selected on the basis of CD4 cell counts, HIV/RNA and clinical status. The Classification and Regression Trees (CART) approach has been used to uncover specific patterns of humoral parameters in different stages of HIV disease. Virus neutralization of primary virus strains and antibodies to gp41 were required to classify patients, suggesting that clinical profiles strongly rely on functional activity against HIV.     

Involvement of Fc gamma RI (CD64) in the mechanism of HIV-1 inhibition by polyclonal IgG purified from infected patients in cultured monocyte-derived macrophages

The aim of this study was to investigate the mechanism of HIV-1 neutralization using monocyte-derived macrophages (MDM) in comparison to PBMC as target cells. For this purpose, we analyzed neutralizing activities of different human polyclonal IgG samples purified from sera of HIV-1-infected individuals using a single cycle infection assay. We found an increase of the neutralizing titer when macrophages vs PBMC were used as target cells. Moreover, polyclonal IgG from HIV-1-infected patients that are not able to neutralize virus when PBMC are used as target cells strongly inhibit MDM infection. Similar results were obtained with neutralizing mAbs. To explore the participation of FcgammaRs in HIV-1 inhibition, F(ab')(2) and Fab of these Igs were produced. Results indicated that both F(ab')(2) and Fab are less effective to inhibit virus replication in MDM. Moreover, competition experiments with Fc fragments of IgG from healthy donors or with purified monoclonal anti-human FcgammaRs Ab strengthen the participation of the FcgammaRs, and in particular of FcgammaRI (CD64) in HIV-1 inhibition on MDM. Mechanisms by which HIV-specific IgG inhibit virus replication in cultured macrophages are proposed and the benefit of inducing such Abs by vaccination is discussed.     

Effect of HIV on antigen presentation by dendritic cells and macrophages

The antigen-presenting function of dendritic cells (DC) and macrophages (MO) following infection with HIV in vitro was examined. Using non-infected cells, DC, but not MO, stimulated primary proliferative responses in allogeneic lymphocytes in the mixed leukocyte reaction. Both DC and MO stimulated secondary responses to influenza virus and to tetanus toxoid in autologous T lymphocytes. After exposure of DC and MO to HIV1 in vitro for 2 days, 27 % of DC but < 1 % MO became infected as assessed by in situ hybridization. DC were blocked in their capacity to stimulate responses to alloantigens or to the recall antigens. By contrast, MO retained the ability to stimulate responses to the recall antigens. Similar effects during in vivo infection would allow activated T-cell clones to respond to antigens presented by MO early in infection. However, any loss of activated T cells might prove cumulative and damaging in the absence of an effective DC recruitment mechanism for resting T cells.     

“Unconventional” neutralizing activity of antibodies against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using “conventional” neutralization assay which uses phytohe-magglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4⁺ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the “conventional” neutralization assay, do exhibit potent and broad neutralizing activity in “unconventional” ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV. Foundation item: Chinese Ministry of Science and Technology 973 program grant awarded to Paul Zhou (2006CB504308).     

Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.     

Antibody-mediated neutralization of primary human immunodeficiency virus type 1 isolates: investigation of the mechanism of inhibition

Human immunodeficiency virus type 1 (HIV-1) neutralization occurs when specific antibodies, mainly those directed against the envelope glycoproteins, inhibit infection, most frequently by preventing the entry of the virus into target cells. However, the precise mechanisms of neutralization remain unclear. Previous studies, mostly with cell lines, have produced conflicting results involving either the inhibition of virus attachment or interference with postbinding events. In this study, we investigated the mechanisms of neutralization by immune sera and compared the inhibition of peripheral blood mononuclear cells (PBMC) infection by HIV-1 primary isolates (PI) with the inhibition of T-cell line infection by T-cell line-adapted (TCLA) strains. We followed the kinetics of neutralization to determine at which step of the viral cycle the antibodies act. We found that neutralization of the TCLA strain HIV-1MN/MT-4 required an interaction between antibodies and cell-free virions before the addition of MT-4 cells, whereas PI were neutralized even after adsorption onto PBMC. In addition, the dose-dependent inhibition of HIV-1MN binding to MT-4 cells was strongly correlated with serum-induced neutralization. In contrast, neutralizing sera did not reduce the adhesion of PI to PBMC. Postbinding inhibition was also detected for HIV-1MN produced by and infecting PBMC, demonstrating that the mechanism of neutralization depends on the target cell used in the assay. Finally, we considered whether the different mechanisms of neutralization may reflect the recognition of qualitatively different epitopes on the surface of PI and HIV-1MN or whether they reflect differences in virus attachment to PBMC and MT-4 cells.     

Opsonization of HIV with complement enhances infection of dendritic cells and viral transfer to CD4 T cells in a CR3 and DC-SIGN-dependent manner

In the present study, we demonstrated that opsonization of primary HIV-1 with human complement enhances infection of immature monocyte-derived dendritic cells (iDC) and transmission in trans of HIV to autologous CD4(+) T lymphocytes. Infection of iDC by opsonized primary R5- and X4-tropic HIV was increased 3- to 5-fold as compared with infection by the corresponding unopsonized HIV. Enhancement of infection was dependent on CR3 as demonstrated by inhibition induced by blocking Abs. The interaction of HIV with CCR5 and CXCR4 on iDC was affected by opsonization. Indeed, stromal-derived factor-1 was more efficient in inhibiting infection of iDC with opsonized R5-tropic HIV-1(BaL) (45%) than with heat-inactivated complement opsonized virus and similarly RANTES inhibited more efficiently infection of iDC with opsonized X4-tropic HIV-1(NDK) (42%) than with heat-inactivated complement opsonized virus. We also showed that attachment of complement-opsonized virus to DC-specific ICAM-grabbing nonintegrin (DC-SIGN) molecule on iDC and HeLa DC-SIGN(+) CR3(-) cells was 46% and 50% higher compared with heat-inactivated complement opsonized virus, respectively. Hence, Abs to DC-SIGN suppressed up to 80% and 60% the binding of opsonized virus to HeLa cells and iDC, respectively. Furthermore, Abs to DC-SIGN inhibited up to 70% of the infection of iDC and up to 65% of infection in trans of autologous lymphocytes with opsonized virus. These results further demonstrated the role of DC-SIGN in complement opsonized virus uptake and infection. Thus, the virus uses complement to its advantage to facilitate early steps leading to infection following mucosal transmission of HIV.     

The effect of HAART on humoral immune response in primary HIV-1 infected patients

The effect of Highly Active Antiretroviral Therapy (HAART) on binding and neutralizing antibody responses to human immunodeficiency virus type-1 (HIV-1) during primary and chronic infection was investigated. Seven patients HAART treated during primary infection, six HAART treated during chronic infection and five patients treated only with ZVD (Zidovudine) were analysed. HAART inhibited the development of anti env antibodies during primary infection. Administering HAART during primary infection usually did not substantially affect the development of weak neutralizing antibody responses against autologous virus. However, we demonstrated that very early treatment, during seroconversion, induce in some cases, a strong neutralizing antibodies against autologous virus. These results may be relevant for understanding how HAART may elicit a strong protective responses and may be useful in developing new strategies designed to achieve a long term control of the HIV infection.     

Restricted occupancy models for neutralization of HIV virions and populations

HIV virions infect cells by attaching to target cell receptors, fusing membranes with the cell and by finally releasing their genetic material into the target cells. Antibodies can hinder the infection by attaching to the HIV envelope glycoprotein trimers before or during attachment. The exact mechanisms and the quantitative requirements of antibody neutralization are still debated. Recently, the number of antibodies rendering one trimer non-functional, called stoichiometry of (trimer) neutralization, was studied with mathematical models. Here we extend this theoretical framework to calculate the stoichiometries of neutralizing a single virion and a whole virion population. We derive mathematical equations for antibody neutralization based on restricted occupancy theory. Additionally we simulate these processes when a direct calculation is not possible. We find that the number of trimers needed for cell entry and the number of antibodies neutralizing one trimer strongly influence the mean number of antibodies needed for virion and population neutralization. Further we show that the mean number of antibodies needed to neutralize a virion population exceeds the product of the number of virions in the population and the mean number of antibodies needed to neutralize one virion.     

Human immunodeficiency virus type 1 variants isolated from single plasma samples display a wide spectrum of neutralization sensitivity

Individuals infected with human immunodeficiency virus type 1 (HIV-1) harbor a mixture of viral variants with different sequences and in some instances with different phenotypic properties. Major and rapid fluctuations in the proportion of viral variants coexisting in an infected individual can be observed under strong pharmacological and immune selective pressure. Because of the short half-life of HIV-infected cells and of HIV virions in the blood, plasma virus populations are highly relevant to HIV evolution in the face of these selective pressures. Here we analyzed the sensitivity to antibody-mediated neutralization of viral variants coexisting in the plasma virus populations of two infected patients. For each patient, several replication-competent viral clones were constructed that carry primary envelope gene sequences obtained from a single plasma sample. Viral clones differed in their tropism and replicative capacity and in the number and positions of glycosylation sites in the envelope glycoproteins. Viruses were tested against heterologous and autologous sera obtained at different time points. Interestingly, we found that viral variants coexisting in each plasma sample were highly heterogeneous in terms of sensitivity to neutralization. The order of sensitivity depended on the serum used and was not associated with virus tropism. The neutralization potency of sera increased with the duration of the infection for both autologous and heterologous neutralization.     

Reversal of human immunodeficiency virus type 1 IIIB to a neutralization-resistant phenotype in an accidentally infected laboratory worker with a progressive clinical course

The role of humoral immunity in controlling human immunodeficiency virus type 1 (HIV-1) is still controversial. The resistance of primary HIV-1 variants to neutralization by antibodies, sera from HIV-1-infected patients, and soluble CD4 protein has been suggested to be a prerequisite for the virus to establish persistence in vivo. To further test this hypothesis, we studied the neutralization sensitivity of two IIIB/LAV variants that were isolated from a laboratory worker who accidentally was infected with the T-cell-line-adapted neutralization-sensitive IIIB isolate. Compared to the original virus in the inoculum, the reisolated viruses showed an increased resistance to neutralization over time. The ratio of nonsynonymous to synonymous nucleotide substitutions in the envelope gene pointed to strong positive selection. The emergence of neutralization-resistant HIV preceded disease development in this laboratory worker. Our results imply that the neutralization resistance of primary HIV may indeed be considered an escape mechanism from humoral immune control.     

Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization

Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed.