A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition.

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Protease inhibitors effectively block cell-to-cell spread of HIV-1 between T cells.

BACKGROUND: The Human Immunodeficiency Virus type-1 (HIV-1) spreads by cell-free diffusion and by direct cell-to-cell transfer, the latter being a significantly more efficient mode of transmission. Recently it has been suggested that cell-to-cell spread may permit ongoing virus replication in the presence of antiretroviral therapy (ART) based on studies performed using Reverse Transcriptase Inhibitors (RTIs). Protease Inhibitors (PIs) constitute an important component of ART; however whether this class of inhibitors can suppress cell-to-cell transfer of HIV-1 is unexplored. Here we have evaluated the inhibitory effect of PIs during cell-to-cell spread of HIV-1 between T lymphocytes. RESULTS: Using quantitative assays in cell line and primary cell systems that directly measure the early steps of HIV-1 infection we find that the PIs Lopinavir and Darunavir are equally potent against both cell-free and cell-to-cell spread of HIV-1. We further show that a protease resistant mutant maintains its resistant phenotype during cell-to-cell spread and is transmitted more efficiently than wild-type virus in the presence of drug. By contrast we find that T cell-T cell spread of HIV-1 is 4-20 fold more resistant to inhibition by the RTIs Nevirapine, Zidovudine and Tenofovir. Notably, varying the ratio of infected and uninfected cells in co-culture impacted on the degree of inhibition, indicating that the relative efficacy of ART is dependent on the multiplicity of infection. CONCLUSIONS: We conclude that if the variable effects of antiviral drugs on cell-to-cell virus dissemination of HIV-1 do indeed impact on viral replication and maintenance of viral reservoirs this is likely to be influenced by the antiviral drug class, since PIs appear particularly effective against both modes of HIV-1 spread.

Relative resistance of HIV-1 founder viruses to control by interferon-alpha.

BACKGROUND: Following mucosal human immunodeficiency virus type 1 (HIV-1) transmission, type 1 interferons (IFNs) are rapidly induced at sites of initial virus replication in the mucosa and draining lymph nodes. However, the role played by IFN-stimulated antiviral activity in restricting HIV-1 replication during the initial stages of infection is not clear. We hypothesized that if type 1 IFNs exert selective pressure on HIV-1 replication in the earliest stages of infection, the founder viruses that succeed in establishing systemic infection would be more IFN-resistant than viruses replicating during chronic infection, when type 1 IFNs are produced at much lower levels. To address this hypothesis, the relative resistance of virus isolates derived from HIV-1-infected individuals during acute and chronic infection to control by type 1 IFNs was analysed. RESULTS: The replication of plasma virus isolates generated from subjects acutely infected with HIV-1 and molecularly cloned founder HIV-1 strains could be reduced but not fully suppressed by type 1 IFNs in vitro. The mean IC50 value for IFNα2 (22 U/ml) was lower than that for IFNß (346 U/ml), although at maximally-inhibitory concentrations both IFN subtypes inhibited virus replication to similar extents. Individual virus isolates exhibited differential susceptibility to inhibition by IFNα2 and IFNß, likely reflecting variation in resistance to differentially up-regulated IFN-stimulated genes. Virus isolates from subjects acutely infected with HIV-1 were significantly more resistant to in vitro control by IFNα than virus isolates generated from the same individuals during chronic, asymptomatic infection. Viral IFN resistance declined rapidly after the acute phase of infection: in five subjects, viruses derived from six-month consensus molecular clones were significantly more sensitive to the antiviral effects of IFNs than the corresponding founder viruses. CONCLUSIONS: The establishment of systemic HIV-1 infection by relatively IFNα-resistant founder viruses lends strong support to the hypothesis that IFNα plays an important role in the control of HIV-1 replication during the earliest stages of infection, prior to systemic viral spread. These findings suggest that it may be possible to harness the antiviral activity of type 1 IFNs in prophylactic and potentially also therapeutic strategies to combat HIV-1 infection.

Potent autologous and heterologous neutralizing antibody responses occur in HIV-2 infection across a broad range of infection outcomes.

Few studies have explored the role of neutralizing antibody (NAb) responses in controlling HIV-2 viremia and disease progression. Using a TZM-bl neutralization assay, we assessed heterologous and autologous NAb responses from a community cohort of HIV-2-infected individuals with a broad range of disease outcomes in rural Guinea-Bissau. All subjects (n = 40) displayed exceptionally high heterologous NAb titers (50% inhibitory plasma dilution or 50% inhibitory concentration [IC(50)], 1:7,000 to 1:1,000,000) against 5 novel primary HIV-2 envelopes and HIV-2 7312A, whereas ROD A and 3 primary envelopes were relatively resistant to neutralization. Most individuals also showed high autologous NAb against contemporaneous envelopes (78% of plasma-envelope combinations in 69 envelopes from 21 subjects), with IC(50)s above 1:10,000. No association between heterologous or autologous NAb titer and greater control of HIV-2 was found. A subset of envelopes was found to be more resistant to neutralization (by plasma and HIV-2 monoclonal antibodies). These envelopes were isolated from individuals with greater intrapatient sequence diversity and were associated with changes in potential N-linked glycosylation sites but not CD4 independence or CXCR4 use. Plasma collected from up to 15 years previously was able to potently neutralize recent autologous envelopes, suggesting a lack of escape from NAb and the persistence of neutralization-sensitive variants over time, despite significant NAb pressure. We conclude that despite the presence of broad and potent NAb responses in HIV-2-infected individuals, these are not the primary forces behind the dichotomous outcomes observed but reveal a limited capacity for adaptive selection and escape from host immunity in HIV-2 infection.

Generation of a family-specific phage library of llama single chain antibody fragments that neutralize HIV-1.

Recently, we described llama antibody fragments (VHH) that can neutralize human immunodeficiency virus, type 1 (HIV-1). These VHH were obtained after selective elution of phages carrying an immune library raised against gp120 of HIV-1 subtype B/C CN54 with soluble CD4. We describe here a new, family-specific approach to obtain the largest possible diversity of related VHH that compete with soluble CD4 for binding to the HIV-1 envelope glycoprotein. The creation of this family-specific library of homologous VHH has enabled us to isolate phages carrying similar nucleotide sequences as the parental VHH. These VHH displayed varying binding affinities and neutralization phenotypes to a panel of different strains and subtypes of HIV-1. Sequence analysis of the homologs showed that the C-terminal three amino acids of the CDR3 loop were crucial in determining the specificity of these VHH for different subtype C HIV-1 strains. There was a positive correlation between affinity of VHH binding to gp120 of HIV-1 IIIB and the breadth of neutralization of diverse HIV-1 envelopes. The family-specific approach has therefore allowed us to better understand the interaction of the CD4-binding site antibodies with virus strain specificity and has potential use for the bioengineering of antibodies and HIV-1 vaccine development.

Extensive complement-dependent enhancement of HIV-1 by autologous non-neutralising antibodies at early stages of infection.

BACKGROUND: Non-neutralising antibodies to the envelope glycoprotein are elicited during acute HIV-1 infection and are abundant throughout the course of disease progression. Although these antibodies appear to have negligible effects on HIV-1 infection when assayed in standard neutralisation assays, they have the potential to exert either inhibitory or enhancing effects through interactions with complement and/or Fc receptors. Here we report that non-neutralising antibodies produced early in response to HIV-1 infection can enhance viral infectivity. RESULTS: We investigated this complement-mediated antibody-dependent enhancement (C'-ADE) of early HIV infection by carrying out longitudinal studies with primary viruses and autologous sera derived sequentially from recently infected individuals, using a T cell line naturally expressing the complement receptor 2 (CR2; CD21). The C'-ADE was consistently observed and in some cases achieved infection-enhancing levels of greater than 350-fold, converting a low-level infection to a highly destructive one. C'-ADE activity declined as a neutralising response to the early virus emerged, but later virus isolates that had escaped the neutralising response demonstrated an increased capacity for enhanced infection by autologous antibodies. Moreover, sera with autologous enhancing activity were capable of C'ADE of heterologous viral isolates, suggesting the targeting of conserved epitopes on the envelope glycoprotein. Ectopic expression of CR2 on cell lines expressing HIV-1 receptors was sufficient to render them sensitive to C'ADE. CONCLUSIONS: Taken together, these results suggest that non-neutralising antibodies to the HIV-1 envelope that arise during acute infection are not 'passive', but in concert with complement and complement receptors may have consequences for HIV-1 dissemination and pathogenesis.

Modulation of HIV-1 macrophage-tropism among R5 envelopes occurs before detection of neutralizing antibodies.

HIV-1 R5 viruses vary widely in their capacity to infect primary macrophages. R5 macrophage-tropism is associated with an increased envelope:CD4 affinity that partly results from an increased exposure of CD4 contact residues on gp120 and allows the use of low levels of CD4 for infection. The selective pressures in vivo that modulate R5 macrophage-tropism are not understood. It is possible that different R5 variants adapt for replication in either T-cells (high CD4) or in macrophages (low CD4). However, other selective pressures in vivo (e.g. neutralizing antibodies) may also impact R5 tropism. Here, we measured macrophage infectivity conferred by gp120 sequences amplified sequentially from subjects in London followed from the acute stage of infection. We report wide variation in the capacity of these envelopes to confer macrophage infection in the complete absence of both autologous and heterologous neutralizing antibodies. Our data show that the variation in macrophage tropism observed at early times cannot have been influenced by neutralizing antibodies.

Novel subtype C human immunodeficiency virus type 1 envelopes cloned directly from plasma: coreceptor usage and neutralization phenotypes.

Human immunodeficiency virus type 1 (HIV-1) is classified into different phylogenetic subtypes, with subtype C representing more than half of the novel infections globally. However, there are relatively few subtype C envelopes available for study. We amplified 18 unique env genes from 13 patients who were infected with subtype C HIV-1 in six African countries and in Scotland to create replication-competent viruses. These envelopes are phylogenetically diverse across the subtype C spectrum, and have on average more N-linked glycosylation sites and slightly longer variable loops than previously described C envelopes. We found that CCR3 coreceptor usage is less prevalent in subtype C than in subtype B viruses, and these envelopes have varied sensitivity to neutralization. The subtype C chimeric viruses generated in this study will be useful for evaluating the breadth of neutralizing antibodies and other entry inhibitors.

Humoral immunity to HIV-1: neutralisation and antibody effector functions.

Several features of HIV have frustrated efforts to develop a vaccine able to induce broadly neutralising antibodies. The enormous genetic diversity of HIV is a major factor, accompanied by the camouflaged nature of the envelope spike, upon which HIV depends for cellular entry and to which antibodies must bind to neutralise. The picture is further complicated by the presence of nonfunctional envelope glycoproteins on the surface of HIV that are immunogenic. Consequently, HIV attracts antibodies that do not directly neutralise the virus but still activate complement and engage Fc receptors, which can both enhance and inhibit infection. The various effects that anti-envelope antibodies have on HIV infection will be reviewed here. Further research is needed to determine if these in vitro-characterised activities have relevance in vivo, and if some of the undesirable effects of non-neutralising antibodies can be avoided or the beneficial effects harnessed.

Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120.

Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B'/C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC(50)) and IC(90) values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.

Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line.

BACKGROUND: Japanese encephalitis virus (JEV), a member of the family Flaviviridae, is an important mosquito-borne human pathogen. Its envelope glycoprotein (E) is the major determinant of the pathogenicity and host immune responses. In the present study, we explored the feasibility of producing recombinant JEV E protein in the virus-free Drosophila expression system. RESULTS: The coding sequence for the signal sequence of premembrane and E protein was cloned into the Drosophila expression vector pAc5.1/V5-His. A Drosophila cell line S2 was cotransfected with this construct as well as a plasmid providing hygromycin B resistance. A cell line expressing the JEV E protein was selected by immunofluoresence, confocal microscopy, and western blot analysis using three different monoclonal antibodies directed against JEV E protein. This cell line was stable in the yield of JEV E protein during two months in vitro maintenance in the presence of hygromycin B. The results showed that the recombinant E protein had an expected molecular weight of about 50 kilodalton, was immunoreactive with all three monoclonal antibodies, and found in both the cytoplasm and culture supernatant. Sucrose gradient ultracentrifugation analysis revealed that the secreted E protein product was in a particulate form. It migrated to the sucrose fraction with a density of 1.13 g/ml. Balb/c mice immunised with the sucrose fraction containing the E protein particles developed specific antibodies. These data show that functioning JEV E protein was expressed in the stable S2 cell line. CONCLUSION: The Drosophila expression system is a more convenient, cheaper and safer approach to the production of vaccine candidates and diagnostic reagents for JEV.

A phase I, randomized study of combined IL-2 and therapeutic immunisation with antiretroviral therapy.

BACKGROUND: Fully functional HIV-1-specific CD8 and CD4 effector T-cell responses are vital to the containment of viral activity and disease progression. These responses are lacking in HIV-1-infected patients with progressive disease. We attempted to augment fully functional HIV-1-specific CD8 and CD4 effector T-cell responses in patients with advanced chronic HIV-1 infection. DESIGN: Chronically infected patients with low CD4 counts T-cell counts who commenced antiretroviral therapy (ART) were subsequently treated with combined interleukin-2 and therapeutic vaccination. METHODS: Thirty six anti-retroviral naive patients were recruited and initiated on combination ART for 17 weeks before randomization to: A) ongoing ART alone; B) ART with IL-2 twice daily for 5 days every four weeks starting at week 17 for 3 cycles; C) ART with IL-2 as in group B and Remune HIV-1 vaccine administered once every 3 months, starting at week 17; and D) ART with Remune vaccine as in group C. Patients were studied for 65 weeks following commencement of ART, with an additional prior 6 week lead-in observation period. CD4 and CD8 T-cell counts, evaluations of HIV-1 RNA levels and proliferative responses to recall and HIV-1 antigens were complemented with assessment of IL-4-secretion alongside quantification of anti-HIV-1 CD8 T-cell responses and neutralizing antibody titres. RESULTS: Neither IL-2 nor Remune vaccination induced sustained HIV-1-specific T-cell responses. However, we report an inverse relationship between HIV-1-specific proliferative responses and IL-4 production which continuously increased in patients receiving immunotherapy, but not patients receiving ART alone. CONCLUSION: Induction of HIV-1-specific cell-mediated responses is a major challenge in chronically HIV-1-infected patients even when combining immunisation with IL-2 therapy. An antigen-specific IL-4-associated suppressive response may play a role in attenuating HIV-specific responses.

Development of the antibody response in acute HIV-1 infection.

BACKGROUND: Cytotoxic T lymphocytes have been shown to reduce viraemia during acute HIV-1 infection; however the role of neutralizing antibodies in this process is unclear. One confounding factor may be artefacts introduced by viral culture. OBJECTIVE: To assess the development of autologous neutralizing and non-neutralizing antibodies following acute HIV-1 infection using recombinant viruses with envelopes amplified directly from patient peripheral blood mononuclear cells, thereby avoiding in vitro selection. METHODS: Disease progression in four homosexual men was monitored from acute infection for up to 2.5 years, in the absence of antiretroviral therapy. Antibodies to viral envelope protein were quantified by enzyme-linked immunosorbent assay. Development of neutralizing antibodies was monitored using a quantitative infectivity reduction assay, sequential serum, recombinant viruses and target cells with defined receptor expression. RESULTS: The time to development of neutralizing antibodies after onset of symptoms was 3, 5, 7 and 16 months in the four patients. There was no correlation between development of neutralizing antibodies and the resolution of viraemia in any of the patients. However, antibodies to the envelope were detectable as early as 2 weeks after onset of symptoms. CONCLUSIONS: Neutralizing antibodies do not contribute to the control of viraemia in acute HIV-1 infection. However, antibodies to the envelope could be detected at the time of reduction in plasma viraemia and so other effector functions of antibodies may play a role in viral clearance.

Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C.

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.

In vivo emergence of HIV-1 highly sensitive to neutralizing antibodies.

BACKGROUND: The rapid and continual viral escape from neutralizing antibodies is well documented in HIV-1 infection. Here we report in vivo emergence of viruses with heightened sensitivity to neutralizing antibodies, sometimes paralleling the development of neutralization escape. METHODOLOGY/PRINCIPAL FINDINGS: Sequential viral envs were amplified from seven HIV-1 infected men monitored from seroconversion up to 5 years after infection. Env-recombinant infectious molecular clones were generated and tested for coreceptor use, macrophage tropism and neutralization sensitivity to homologous and heterologous serum, soluble CD4 and monoclonal antibodies IgG1b12, 2G12 and 17b. We found that HIV-1 evolves sensitivity to contemporaneous neutralizing antibodies during infection. Neutralization sensitive viruses grow out even when potent autologous neutralizing antibodies are present in patient serum. Increased sensitivity to neutralization was associated with susceptibility of the CD4 binding site or epitopes induced after CD4 binding, and mediated by complex envelope determinants including V3 and V4 residues. The development of neutralization sensitive viruses occurred without clinical progression, coreceptor switch or change in tropism for primary macrophages. CONCLUSIONS: We propose that an interplay of selective forces for greater virus replication efficiency without the need to resist neutralizing antibodies in a compartment protected from immune surveillance may explain the temporal course described here for the in vivo emergence of HIV-1 isolates with high sensitivity to neutralizing antibodies.

Mapping the immune response to the outer domain of a human immunodeficiency virus-1 clade C gp120.

The outer domain (OD) of human immunodeficiency virus (HIV)-1 gp120 represents an attractive, if difficult, target for a beneficial immune response to HIV infection. Unlike the entire gp120, the OD is structurally stable and contains the surfaces that interact with both the primary and secondary cellular receptors. The primary strain-specific neutralizing target, the V3 loop, lies within the OD, as do epitopes for two cross-reactive neutralizing monoclonal antibodies (mAbs), b12 and 2G12, and the contact sites for a number of inhibitory lectins. The OD is poorly immunogenic, at least in the context of complete gp120, but purposeful OD immunization can lead to a substantial antibody response. Here, we map the antibody generated following immunization with a clade C OD. In contrast to published data for the clade B OD, the majority of the polyclonal response to the complete clade C OD is to the V3 loop; deletion of the loop substantially reduces immunogenicity. When the loop sequence was substituted for the epitope for 2F5, a well-characterized human cross-neutralizing mAb, a polyclonal response to the epitope was generated. A panel of mAbs against the clade C OD identified two mAbs that reacted with the loop and were neutralizing for clade C but not B isolates. Other mAbs recognized both linear and conformational epitopes in the OD. We conclude that, as for complete gp120, V3 immunodominance is a property of OD immunogens, that the responses can be neutralizing and that it could be exploited for the presentation of other epitopes.

Clade specific neutralising vaccines for HIV: an appropriate target?

The enormous diversity of the human immunodeficiency virus (HIV) has led to the idea that designing vaccines to specific geographic regions, or clades, could simplify the complexity of the task. Yet, despite the sequence diversity, all HIV viruses known to date interact with the same cellular receptors (CD4 and/or a coreceptor, CCR5 or CXCR4). In this review we examine the existing evidence to support a clade-specific vaccine strategy for induction of neutralising antibodies. We concentrate on lessons learnt from natural infection of humans. In short, the vast majority of studies to date indicate that neutralisation of HIV-1 is not clade specific. Potent sera tend to neutralise a range of heterologous viruses with no apparent clade preference, and none of the human neutralising monoclonal antibodies so far generated demonstrate significant clade preference. All but one of the most broadly neutralising antibodies are to functional regions involved in receptor interactions and plasma membrane fusion. Given these facts, we suggest that vaccine approaches that focus on 'clade-specific' and 'clade-generic' vaccines will logically converge on the same functionally conserved envelope structures. It still remains to be determined whether or not the task of designing a 'clade-generic' vaccine could be simplified by focusing on the viral envelopes with 'transmitting phenotypes'.

Novel envelope determinants for CCR3 use by human immunodeficiency virus.

Human immunodeficiency virus type 1 can generally use CCR3 and CCR5 for cell entry. We show that envelopes with novel phenotypes arise during "coreceptor switch": one loses the ability to use CCR3 (R5-only phenotype), and another gains use of CXCR4 in addition to CCR5 and CCR3 (R3/R5/X4-using phenotype). The envelope determinants for CCR3 use mapped to three amino acids. One, N356 in conserved region 3, is a potential glycosylation site and has not previously been associated with coreceptor use. The other two, R440 and N448 in conserved region 4, are proximal to but distinct from residues already identified as being important for CCR5 binding.

Primary CCR5 only using HIV-1 isolates does not accurately represent the in vivo replicating quasi-species.

Most HIV-1 isolates depend on CCR5 or CXCR4 to infect target cells, and efficient use of other coreceptors is rare. We cloned HIV-1 envelopes from virus at acute infection and found that most use CCR3 efficiently. This result contradicts prevailing data, suggesting that CCR3 usage is rare. We hypothesized that direct isolation into PBMC biases selection of viruses that use CCR5 and not CCR3. We therefore compared coreceptor use of isolates obtained by PBMC coculture with envelopes cloned directly from patient blood samples, which should represent actively replicating species. Viruses derived by cloning generally used CCR3 and CCR5 with equally efficiently. In contrast, we found that viruses isolated by PBMC coculture largely, or exclusively, used CCR5. Regardless of whether CCR3 use contributes to HIV-1 transmission or pathogenesis, our results demonstrate that "primary isolates" generated by PBMC culture are unlikely to accurately represent the in vivo replicating quasi-species.

Detection of antibody-dependent complement-mediated inactivation of both autologous and heterologous virus in primary human immunodeficiency virus type 1 infection.

Specific CD8 T-cell responses to human immunodeficiency virus type 1 (HIV-1) are induced in primary infection and make an important contribution to the control of early viral replication. The importance of neutralizing antibodies in containing primary viremia is questioned because they usually arise much later. Nevertheless antienvelope antibodies develop simultaneously with, or even before, peak viremia. We determined whether such antibodies might control viremia by complement-mediated inactivation (CMI). In each of seven patients studied, antibodies capable of CMI appeared at or shortly after the peak in viremia, concomitantly with detection of virus-specific T-cell responses. The CMI was effective on both autologous and heterologous HIV-1 isolates. Activation of the classical pathway and direct viral lysis were at least partly responsible. Since immunoglobulin G (IgG)-antibodies triggered the CMI, specific memory B cells could also be induced by vaccination. Thus, consideration should be given to vaccination strategies that induce IgG antibodies capable of CMI.