Neutralization Serotyping of BK Polyomavirus Infection in Kidney Transplant Recipients

BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy.     

Evaluation and analysis of dengue virus enhancing and neutralizing activities using simple high-throughput assays

The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose–response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.     

High-Avidity and Potently Neutralizing Cross-Reactive Human Monoclonal Antibodies Derived from Secondary Dengue Virus Infection

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination.     

Human immunodeficiency virus neutralizing antibodies recognize several conserved domains on the envelope glycoproteins.

Serum neutralizing antibodies against the human immunodeficiency virus were frequently detected in infected individuals, and low or absent serum neutralizing titers correlated with poor prognosis. Multiple diverse human immunodeficiency virus isolates were found to exhibit similar susceptibility to neutralization by a panel of human seropositive sera, suggesting that neutralizing antibodies are largely directed against conserved viral domains. Furthermore, utilizing antisera raised against a library of synthetic env peptides, four regions which are important in the neutralization process have been identified within both human immunodeficiency virus envelope glycoproteins (gp41 and gp120). Three of these are in conserved domains and should be considered for inclusion in a candidate vaccine.     

The Type-Specific Neutralizing Antibody Response Elicited by a Dengue Vaccine Candidate Is Focused on Two Amino Acids of the Envelope Protein

Dengue viruses are mosquito-borne flaviviruses that circulate in nature as four distinct serotypes (DENV1-4). These emerging pathogens are responsible for more than 100 million human infections annually. Severe clinical manifestations of disease are predominantly associated with a secondary infection by a heterotypic DENV serotype. The increased risk of severe disease in DENV-sensitized populations significantly complicates vaccine development, as a vaccine must simultaneously confer protection against all four DENV serotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of ongoing vaccine development efforts. However, a recent large clinical trial of a candidate live-attenuated DENV vaccine revealed low protective efficacy despite eliciting a neutralizing antibody response, highlighting the need for a better understanding of the humoral immune response against dengue infection. In this study, we sought to identify epitopes recognized by serotype-specific neutralizing antibodies elicited by monovalent DENV1 vaccination. We constructed a panel of over 50 DENV1 structural gene variants containing substitutions at surface-accessible residues of the envelope (E) protein to match the corresponding DENV2 sequence. Amino acids that contribute to recognition by serotype-specific neutralizing antibodies were identified as DENV mutants with reduced sensitivity to neutralization by DENV1 immune sera, but not cross-reactive neutralizing antibodies elicited by DENV2 vaccination. We identified two mutations (E126K and E157K) that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. Longitudinal and cross-sectional analysis of sera from 24 participants of a phase I clinical study revealed a markedly reduced capacity to neutralize a E126K/E157K DENV1 variant. Sera from 77% of subjects recognized the E126K/E157K DENV1 variant and DENV2 equivalently (<3-fold difference). These data indicate the type-specific component of the DENV1 neutralizing antibody response to vaccination is strikingly focused on just two amino acids of the E protein. This study provides an important step towards deconvoluting the functional complexity of DENV serology following vaccination.     

In-depth analysis of the antibody response of individuals exposed to primary dengue virus infection

Humans who experience a primary dengue virus (DENV) infection develop antibodies that preferentially neutralize the homologous serotype responsible for infection. Affected individuals also generate cross-reactive antibodies against heterologous DENV serotypes, which are non-neutralizing. Dengue cross-reactive, non-neutralizing antibodies can enhance infection of Fc receptor bearing cells and, potentially, exacerbate disease. The actual binding sites of human antibody on the DENV particle are not well defined. We characterized the specificity and neutralization potency of polyclonal serum antibodies and memory B-cell derived monoclonal antibodies (hMAbs) from 2 individuals exposed to primary DENV infections. Most DENV-specific hMAbs were serotype cross-reactive and weakly neutralizing. Moreover, many hMAbs bound to the viral pre-membrane protein and other sites on the virus that were not preserved when the viral envelope protein was produced as a soluble, recombinant antigen (rE protein). Nonetheless, by modifying the screening procedure to detect rare antibodies that bound to rE, we were able to isolate and map human antibodies that strongly neutralized the homologous serotype of DENV. Our MAbs results indicate that, in these two individuals exposed to primary DENV infections, a small fraction of the total antibody response was responsible for virus neutralization.     

Neutralization of respiratory syncytial virus after cell attachment.

Little is known about the mechanisms of antibody-mediated neutralization of respiratory syncytial virus (RSV) which causes recurrent infections in human despite the virtually universal presence of neutralizing serum antibodies. Human serum neutralization titers showed strong correlation with post-cell-attachment neutralizing titers for both RSV-convalescent sera and control sera but showed less strong correlation with cell-attachment blocking titers. Neutralization was effective for the first 60 min of infection, indicating that immune serum-mediated neutralization of RSV infection largely involves inhibition of early events following cell attachment.     

Human monoclonal antibodies to neutralize all dengue virus serotypes using lymphocytes from patients at acute phase of the secondary infection

The global spread of the four dengue virus serotypes (DENV-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, we prepared hybridomas producing human monoclonal antibodies (HuMAbs) against DENV using peripheral blood mononuclear cells (PBMCs) from patients in the acute phase (around 1 week after the onset of illness) or the convalescent phase (around 2weeks after the onset of illness) of secondary infection. Interestingly, a larger number of hybridoma clones was obtained from patients in the acute phase than from those in the convalescent phase. Most HuMAbs from acute-phase infections were cross-reactive with all four DENV serotypes and showed significant neutralization activity to all four DENV serotypes. Thus, secondary DENV infection plays a significant role in stimulating memory cells to transiently increase the number of antibody-secreting plasma cells in patients in the early phase after the secondary infection. These HuMAbs will enable us to better understand the protective and pathogenic effects of DENV infection, which could vary greatly among secondarily-infected individuals.     

Analysis of Cross-Reactive Antibodies Recognizing the Fusion Loop of Envelope Protein and Correlation with Neutralizing Antibody Titers in Nicaraguan Dengue Cases

Dengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. The envelope (E) protein of DENV is the major target of neutralizing antibodies (Abs). Previous studies have shown that a significant proportion of anti-E Abs in human serum after DENV infection recognize the highly conserved fusion loop (FL) of E protein. The role of anti-FL Abs in protection against subsequent DENV infection versus pathogenesis remains unclear. A human anti-E monoclonal Ab was used as a standard in a virion-capture ELISA to measure the concentration of anti-E Abs, [anti-E Abs], in dengue-immune sera from Nicaraguan patients collected 3, 6, 12 and 18 months post-infection. The proportion of anti-FL Abs was determined by capture ELISA using virus-like particles containing mutations in FL, and the concentration of anti-FL Abs, [anti-FL Abs], was calculated. Neutralization titers (NT₅₀) were determined using a previously described flow cytometry-based assay. Analysis of sequential samples from 10 dengue patients revealed [anti-E Abs] and [anti-FL Abs] were higher in secondary than in primary DENV infections. While [anti-FL Abs] did not correlate with NT₅₀ against the current infecting serotype, it correlated with NT₅₀ against the serotypes to which patients had likely not yet been exposed (“non-exposed” serotypes) in 14 secondary DENV3 and 15 secondary DENV2 cases. These findings demonstrate the kinetics of anti-FL Abs and provide evidence that anti-FL Abs play a protective role against “non-exposed” serotypes after secondary DENV infection.     

Protection against High-Dose Highly Pathogenic Mucosal SIV Challenge at Very Low Serum Neutralizing Titers of the Antibody-Like Molecule CD4-IgG2

Passive transfer studies using monoclonal or polyclonal antibodies in the macaque model have been valuable for determining conditions for antibody protection against immunodeficiency virus challenge. Most studies have employed hybrid simian/human immunodeficiency virus (SHIV) challenge in conjunction with neutralizing human monoclonal antibodies. Passive protection against SIV, particularly the pathogenic prototype virus SIVmac239, has been little studied because of the paucity of neutralizing antibodies to this virus. Here, we show that the antibody-like molecule CD4-IgG2 potently neutralizes SIVmac239 in vitro. When administered by an osmotic pump to maintain concentrations given the short half-life of CD4-IgG2 in macaques, the molecule provided sterilizing immunity/protection against high-dose mucosal viral challenge to a high proportion of animals (5/7 at a 200 mg dose CD4-IgG2 and 3/6 at a 20 mg dose) at serum concentrations below 1.5 μg/ml. The neutralizing titers of such sera were predicted to be very low and indeed sera at a 1∶4 dilution produced no neutralization in a pseudovirus assay. Macaque anti-human CD4 titers did develop weakly at later time points in some animals but were not associated with the level of protection against viral challenge. The results show that, although SIVmac239 is considered a highly pathogenic virus for which vaccine-induced T cell responses in particular have provided limited benefit against high dose challenge, the antibody-like CD4-IgG2 molecule at surprisingly low serum concentration affords sterilizing immunity/protection to a majority of animals.     

Neutralizing antibodies to an immunodominant envelope sequence do not prevent gp120 binding to CD4.

Animals immunized with the human immunodeficiency virus type 1 gp160 glycoprotein or certain recombinant envelope components develop potent virus-neutralizing activity. This activity is principally due to antibodies directed toward a hypervariable region of gp120 between cysteine residues 302 and 337 and is virus isolate specific. These antisera, as well as two neutralizing monoclonal antibodies directed against the same hypervariable sequence, do not appreciably block gp120 from binding CD4. In contrast, serum samples from infected humans possess high titers of antibodies that block gp120-CD4 binding; these titers approximately correlate with the serum neutralization titers. Our results suggest that there are at least two targets on the envelope glycoprotein for virus neutralization. The target responsible for the broader neutralizing activity of human serum may be a conserved region of gp120 involved in CD4 binding. The antibodies directed at the hypervariable region of the envelope inhibit a different step in virus infection which is subsequent to receptor binding. The extent to which these two different epitopes of gp120 may be involved in protection against human immunodeficiency virus infection is discussed.     

Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II

The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.     

Strain-Specific Neutralization of Human Cytomegalovirus Isolates by Human Sera

Induction of an effective antibody response against human cytomegalovirus (HCMV) is an important defense mechanism since it is potentially capable of neutralizing infectious viruses. We have analyzed the extent of HCMV strain-specific neutralization capacity in human sera. Nine recent HCMV isolates and their corresponding sera were investigated in cross-neutralization assays. We observed differences, independent of the overall neutralization capacity, in the 50% neutralization titers of the sera against individual strains, differences that ranged from 8-fold to more than 60-fold. For one isolate, complete resistance to neutralization by two human sera was observed. The neutralization capacity of human sera was not influenced by the presence of various concentrations (up to 100-fold excess) of noninfectious envelope glycoproteins, an inherent contamination of virus preparations from recent HCMV isolates. This indicated that the decisive parameter for neutralization is the titer of the neutralizing antibodies and that neutralization is largely independent of the concentration of virus. Analysis with transplant patients revealed that during primary infection strain-specific and strain-common antibodies are produced asynchronously. Thus, our data demonstrate that the induction of strain-specific neutralizing antibodies is a common event during infection with HCMV and that it might have important implications for the course of the infection and the development of anti-HCMV vaccines.     

The ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region

Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162DeltaV2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840-7845, 1998). This observation led us to propose that the modified, SF162DeltaV2-derived envelope may elicit higher titers of cross-reactive neutralizing antibodies than the unmodified SF162-derived envelope. To test this hypothesis, we immunized rabbits and rhesus macaques with the gp140 form of these two envelopes. In rabbits, both immunogens elicited similar titers of binding antibodies but the modified immunogen was more effective in eliciting neutralizing antibodies, not only against the SF162DeltaV2 and SF162 viruses but also against several heterologous primary HIV type 1 (HIV-1) isolates. In rhesus macaques both immunogens elicited potent binding antibodies, but again the modified immunogen was more effective in eliciting the generation of neutralizing antibodies against the SF162DeltaV2 and SF162 viruses. Antibodies capable of neutralizing several, but not all, heterologous primary HIV-1 isolates tested were elicited only in macaques immunized with the modified immunogen. The efficiency of neutralization of these heterologous isolates was lower than that recorded against the SF162 isolate. Our results strongly suggest that although soluble oligomeric envelope subunit vaccines may elicit neutralizing antibody responses against heterologous primary HIV-1 isolates, these responses will not be broad and potent unless specific modifications are introduced to increase the exposure of conserved neutralization epitopes.     

Generation of Monoclonal Antibodies against Dengue Virus Type 4 and Identification of Enhancing Epitopes on Envelope Protein

The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.     

Comparison of infection-neutralizing and -enhancing antibody balance induced by two distinct genotype strains of dengue virus type 1 or 3 DNA vaccines in mice

Dengue viruses have spread throughout tropical and subtropical countries, and vaccine development is urgently needed. However, one concern is that induction of insufficient levels of neutralizing antibodies in vaccines may increase disease severity because of a hypothetical mechanism termed antibody-dependent enhancement of infection. This study used two distinct genotype strains of dengue virus types 1 and 3 (DENV1 and DENV3, respectively) to compare antibody responses in a mouse-DNA vaccine model. As expected, a conventional neutralization test using Vero cells showed higher antibody titers in homologous rather than heterologous combinations of genotype strains used for mouse immunization and the neutralization test, for each of DENV1 and DENV3. However, our assay system using K562 cells to measure the balance of neutralizing and enhancing antibodies indicated that Vero cell-neutralizing antibody titers did not always correlate with enhancing activities observed at subneutralizing doses. Rather, induction of enhancing activities depended on the genotype strain used for mouse immunization. The genotype/strain difference also affected IgG subclass profiles and potentially the composition of antibody species induced in mice. This study suggests that enhancing activities of dengue virus-induced neutralizing antibodies may vary according to the genotype and has implications for vaccine antigen development.     

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.     

Human immunodeficiency virus (HIV)-positive sera obtained shortly after seroconversion neutralize autologous HIV type 1 isolates on primary macrophages but not on lymphocytes

The aim of this study was to analyze the role of humoral immunity in early human immunodeficiency virus (HIV) infection. As neutralizing activities in HIV-positive sera are rarely detectable earlier than 9 to 12 months after infection using primary lymphocytes as target cells in neutralization assays, humoral immunity is generally thought not to contribute significantly to early virus control in the patients. Besides lymphocytes, cells of the monocyte/macrophage lineage are known to be important target cells for HIV in vivo during the establishment of the infection. Therefore, we studied the neutralization of early primary HIV isolates by autologous serum samples using primary macrophages as target cells in the neutralization assays. We analyzed neutralizing activities against the autologous HIV-1 isolates in 10 patients' sera taken shortly after seroconversion, both on primary macrophages and, for comparison, on lymphocytes. Viruses were isolated and expanded in primary mixed cultures containing macrophages and lymphocytes in order to avoid selection for one particular cell type. All viruses replicated to different degrees in macrophages and lymphocytes; nine had a nonsyncytium-inducing phenotype, and one was syncytium inducing. The detection of neutralizing antibodies in acute primary HIV infection depended on the target cells used. Confirming previous studies, we did not find neutralizing activities on lymphocytes at this early time point. In contrast, neutralizing activities were detectable in the same sera if primary macrophages were used as target cells. Differences in neutralizing activities on macrophages and lymphocytes were not due to different virus variants being present in the different cell systems, as gp120 sequences derived from both cell types were homogeneous. Neutralization activities on macrophages did not correlate with the amount of beta-chemokines in the sera. As affinity-purified immunoglobulin G preparations from an early patient serum also exhibited neutralization of the autologous virus isolate on primary macrophages, but not on lymphocytes, neutralization is very likely due to antibodies against viral epitopes necessary for infection of macrophages but not for infection of lymphocytes. Our data suggest that, along with cell-mediated immunity, humoral immunity may contribute to the reduction of primary viremia in the patient. This was further supported by a certain association between neutralizing antibody titers on macrophages and viral load in the patients.     

Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.     

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.