Epitope-specific antibody responses to virulent and avirulent feline infectious peritonitis virus isolates.

Feline infectious peritonitis virus (FIPV) has been isolated several times from infected cats. Some of these isolates vary markedly in their ability to cause disease. Specific-pathogen-free cats were inoculated with the avirulent FIPV-UCD-2 isolate or the extremely virulent FIPV-79-1146 isolate or both. After 1 month, cats which had received FIPV-79-1146 were either dead or showed clinical signs of FIP. All cats which received only FIPV-UCD-2 remained healthy up to 6 months after inoculation. Antibody-mediated immune enhancement of disease was not observed in cats which received FIPV-UCD-2 before inoculation with FIPV-79-1146. Monoclonal antibodies which recognized type-specific epitopes on each of the structural polypeptides of these two viruses were used in competitive-inhibition enzyme-linked immunosorbent assays to analyze the humoral immune responses of the cats. All cats produced antibodies to epitopes found on the homologous virus. In addition, cats inoculated with FIPV-79-1146 also produced antibodies which inhibited the binding of the anti-FIPV-UCD-2 E1 monoclonal antibody. One cat inoculated twice with FIPV-UCD-2 produced antibodies which inhibited the binding of the anti-FIPV-79-1146 N- and E1-specific monoclonal antibodies. Competitive enzyme-linked immunosorbent assays may prove useful in distinguishing cats which are infected with virulent FIPV isolates from cats infected with avirulent feline coronaviruses.     

Heterogeneity of human immunodeficiency virus cell-associated antigens and demonstration of virus type specificities of human antibody responses

We examined the antigens of human immunodeficiency viruses (HIV) expressed on infected H9 cells using live-cell membrane immunofluorescence and immunofluorescence absorption. Application of this nondenaturing serological method permitted analysis of HIV antigenic determinants maintained in their native configurations on the cell surface. Sera from infected individuals were found to react variably with H9 cells productively infected with nine different HIV isolates, and certain sera were completely unreactive with some isolates. Absorption of the sera prior to use in immunofluorescence revealed extensive heterogeneity of HIV cell-surface antigens and multiple type-specific antibodies in patients' sera. Immunoprecipitation and SDS-PAGE analysis of radiolabeled cell-surface proteins indicated that the predominant serological reactions were to env-encoded proteins. The observed antigenic and antibody heterogeneity likely reflects env sequence heterogeneity which has been previously reported for different HIV isolates. The demonstration of antigenic diversity among HIVs and the importance of defining the native antigenic epitopes, particularly those most widely shared, are important issues that must be considered in vaccine development.     

Evaluation of passive particle agglutination test for antibody to human immunodeficiency virus.

A gelatin particle agglutination assay was compared with indirect immunofluorescence by using 663 serum samples from acquired immunodeficiency syndrome and acquired immunodeficiency syndrome-related complex patients and asymptomatic male homosexuals in the United States and from hemophiliacs and healthy adult controls in Japan. The results showed that all 104 samples which were positive by indirect immunofluorescence were also positive by particle agglutination, while 5 additional samples were positive by particle agglutination only. The coincidence rate for antibody-positive and antibody-negative specimens was 99% (658 of 663) between particle agglutination and immunofluorescence. Four of the five samples which were positive by particle agglutination only were found by radioimmunoprecipitation to contain anti-env gene products of human immunodeficiency virus. Antibody titers of samples giving a positive reaction by particular agglutination varied from low (titer, 256) to remarkably high (256 X 10(5)). All specimens having particle agglutination titers of more than 10(5) were positive by immunofluorescence. A high correlation (r = 0.66) was observed between the titers of antibodies determined by particle agglutination and those determined by immunofluorescence. After fractionation of a serum sample from an individual at high risk by using high-performance liquid chromatography, it was shown that immunoglobulin M as well as immunoglobulin G human immunodeficiency virus antibody was detected by particle agglutination. Additional serum samples with a potential risk of giving false-positive results, such as heat-treated specimens, specimens containing antibodies to HLA, specimens containing auto-antibodies, and serum samples from individuals with a history of multiple blood transfusions, were shown to be clearly negative by particle agglutination.     

Standardized microtiter assay for determination of syncytium-inducing phenotypes of clinical human immunodeficiency virus type 1 isolates.

A standardized assay in 96-well microtiter plates for syncytium-inducing (SI) human immunodeficiency virus type 1 phenotype detection using MT-2 cells has been developed. SI variants were found in 67% of the patients with advanced human immunodeficiency virus disease. The occurrence of the SI phenotype increased with lower CD4+ counts. There was no association between p24 antigenemia and the SI phenotype.     

Neutralizing antibody responses to varicella-zoster virus.

Neutralization of varicella-zoster (V-Z) virus by human sera and immune rhesus monkey sera was enhanced by fresh guinea pig complement. There was no marked difference in the degree to which complement enhanced neutralization by sera from current V-Z virus infections and sera from long-past varicella infections. Immunoglobulin G neutralizing antibody in sera from varicella cases was enhanced by complement to a slightly higher degree than was immunoglobulin M (IgM) antibody, and immunoglobulin G neutralizing antibody in immune monkey sera was enhanced to a much greater degree than was IgM antibody. There was a rapid decline in the complement requirement of IgM neutralizing antibodies over the course of immunization of the rhesus monkeys. V-Z neutralizing antibody titers in the presence of complement were higher than complement-fixing titers of the same sera in all groups of individuals studied. IgM neutralizing antibody for V-Z virus was demonstrable in all cases of varicella but in only 1 of 22 zoster cases, and V-Z IgM neutralizing antibody was not detectable in primary herpes simplex virus infections in which heterotypic antibody titer rises occurred to V-Z virus. Complement-fixing antibody for V-Z virus was absent in 19S serum fractions which contained IgM neutralizing antibody for the virus.     

A sensitive assay for detection and measurement of neutralising antibody to human immunodeficiency virus.

An assay based on the inhibition of syncytium formation in C8166 cells was developed to measure low levels of neutralising antibody (NT-AB) to human immunodeficiency virus (HIV) and to detect cross-reactivity between virus strains. The relationship between virus challenge and antibody titre was represented by a tripartite curve which was essentially linear over moderate levels of virus input. Based on these findings, antibody titres were standardised against 100 TCID50 of challenge virus. However, lower virus inocula were found to detect minimum levels of antibody. Reproducibility of antibody titres between tests was high, with variation generally lying within one dilution step. The improved sensitivity of the technique allowed detection of NT-ABs in animals immunised with immune-stimulating complexes (ISCOMS) incorporating HIV antigens. Consistent levels of cross-reactivity between HIV strains was demonstrated, indicating the presence of distinct viral groups, from which dominant isolates may be chosen for use in vaccination studies.     

Accentuated antibody response to paramyxoviruses in individuals infected with human immunodeficiency virus

Sera from 31 human immunodeficiency virus (HIV)-infected patients, representing different clinical stages of HIV infection, were assayed for antibodies against measles and mumps viruses by various serological tests and compared to 23 healthy controls. Sera from four patients (two primary, one asymptomatic, and one acquired immunodeficiency syndrome) exhibited a pronounced antibody response to measles as detected by haemagglutination inhibition and radioimmuno-precipitation assay. The RIPA-positive sera showed increased reactivity to all the viral components and in particular to the haemagglutinin (HA) protein of the virus (Fig. 1). Three of these positive patients also showed a similar response to mumps virus. One of the control sera also showed an increase in antibody titre in measles serological tests. The measles antibodies were shown not be anti-HIV antibodies crossreacting with paramyxoviruses. The reactivity to haemagglutinin was still present when using nonglycosylated measles virus antigen grown in the presence of tunicamycin. Whether the accentuated antibody response is due to polyclonal activation mediated by HIV or to reactivation of the viruses remains to be answered.     

Patterns of antigenaemia and antibody response in patients infected with human immunodeficiency virus (HIV) according to clinical state.

Five hundred and fifty six subjects, known to be homosexuals or intravenous drug abusers and seropositive for HIV antibody, were selected on the basis of their clinical state--symptom free, lymphadenopathy syndrome (LAS), AIDS related complex (ARC), and AIDS. The presence of antigenaemia and the humoral response to viral polypeptides was investigated. The prevalence of patients positive for p31 antibody was significantly increased in those with AIDS and detectable antigenaemia.     

Evaluation of two commercially available anti human immunodeficiency virus antibody ELISA Kits using clinical samples

Laboratory diagnosis is the mainstay in the diagnosis of HIV infection in an individual. The wide range of diagnostic kits available, for the detection of anti HIV antibodies, makes it mandatory that the kits are evaluated before they are made available in the market. We evaluated the performance of a Microlisa HIV kit (J. Mitra & Co.) using a panel of HIV reactive and non reactive clinical specimens. The sensitivity and specificity of the kit were found to be 100% as compared to the UBI HIV 1/2 EIA kit. The ease of testing, the assay characteristics including efficiency of the Microlisa favour its use by laboratories as a primary screen for HIV infection.     

Enzyme immunoassay using a novel recombinant polypeptide to detect human immunodeficiency virus env antibody.

A unique antigen, CBre3, has been synthesized from a genetically engineered clone to detect human immunodeficiency virus (HIV) env antibodies with high sensitivity and specificity. The antigen contains sequences derived from both envelope proteins of HIV, i.e., gp120 and gp41, and was purified free of Escherichia coli proteins detectable by Coomassie stain or immunoblotting with E. coli antiserum. The purified recombinant polypeptides were used as antigen in an enzyme immunoassay (EIA) to screen serum samples from healthy and HIV-infected individuals. The same samples were also tested by radioimmunoprecipitation (RIP) for gp120 and gp160 HIV antibodies. All samples containing gp120 and gp160 antibodies by RIP had CBre3 EIA values greater than 0.35 (n, 122; range, 0.37 to 2.1+; median, 1.65). All RIP HIV antibody-negative samples had CBre3 EIA values less than 0.25 (n, 140; mean, 0.052; standard deviation, 0.045; range, 0.00 to 0.22). The endpoint titer of a standard positive control serum was 1:10,000 by RIP and by CBre3 EIA. The assay was 100% accurate in three proficiency panels. It easily detected six samples from individuals whose infections were confirmed by culture; these samples were reactive only with p24 by Western blot. The samples also were positive for gp120 and gp160 antibodies by RIP. These data suggest that the CBre3 EIA can detect env antibodies as sensitively and specifically as RIP and with more sensitivity than Western blot.     

Co-evolution of human immunodeficiency virus and cytotoxic T-lymphocyte responses

After more than a decade of intensive research, the precise of human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) in determining the course of the infection remains open to argument. It is established that HIV-specific CTL appear early in the infection and are temporally associated with the clearance of culturable virus from the blood; that CTL are generally detectable at very high levels throughout the asymptomatic phase and decline at the time of progression to AIDS; and that CTL-mediated killing is sufficiently fast to prevent production of new virus by HIV-infected cells. However, viral turnover is throughout the course of the infection, and –infected individuals progress inexorably to disease in spite of the CTL response. In order Co address the question of whether CTL play an active part in influencing the course of HIV infection, one approach has been to seek evidence for CTL-mediated selection pressure on the virus. Several clear examples of CTL epitope-specific mutations selected to fixation are described. We argue that CTL escape is a common event which occurs at all stages of the Infection. Detailed longitudinal studies are required to detect CTL escape and to nd the complexities contributed by factors SUC h as a polyvalent CTL response and the presence of epitope variants which antagonise the CTL response. In conclusion, there is strong evidence of a dynamic process in which CTL impose important selection constraints upon HIV fro m which the virus attempts to escape; ultimately, at the time of disease progression, the tenuous control of CTL over the virus is lost.     

Cytotoxic T lymphocyte responses to human immunodeficiency virus: control and escape

Effective preventive and therapeutic intervention in individuals exposed to or infected with human immunodeficiency virus (HIV) depends, in part, on a clear understanding of the interactions between the virus and those elements of the host immune response which control viral replication. Recent advances have provided compelling evidence that cytotoxic T lymphocytes (CTLs) constitute an essential component of protective antiretroviral immunity. Here, we review briefly the significance of this work in the context of previous studies, and outline the mechanisms through which HIV evades CTL activity.     

Epitopes for broad and potent neutralizing antibody responses during chronic infection with human immunodeficiency virus type 1

Neutralizing antibody (nAb) response is sporadic and has limited potency and breadth during infection with human immunodeficiency virus type 1 (HIV-1). In rare cases, broad and potent nAbs are actually induced in vivo. Identifying specific epitopes targeted by such broad and potent nAb response is valuable in guiding the design of a prophylactic vaccine aimed to induce nAb. In this study, we have defined neutralizing epitope usage in 7 out of 17 subjects with broad and potent nAbs by using targeted mutagenesis in known neutralizing epitopes of HIV-1 glycoproteins and by using in vitro depletion of serum neutralizing activity by various recombinant HIV-1 glycoproteins. Consistent with recent reports, the CD4 binding site (CD4BS) is targeted by nAbs in vivo (4 of the 7 subjects with defined neutralizing epitopes). The new finding from this study is that epitopes in the gp120 outer domain are also targeted by nAbs in vivo (5 of the 7 subjects). The outer domain epitopes include glycan-dependent epitopes (2 subjects), conserved nonlinear epitope in the V3 region (2 subjects), and a CD4BS epitope composed mainly of the elements in the outer domain (1 subject). Importantly, we found indication for epitope poly-specificity, a dual usage of the V3 and CD4BS epitopes, in only one subject. This study provides a more complete profile of epitope usage for broad and potent nAb responses during HIV-1 infection.     

Antibody responses to Haemophilus influenzae type B vaccines in men with human immunodeficiency virus infection.

BACKGROUND. Persons with human immunodeficiency virus (HIV) infection are at increased risk for serious infections caused by Haemophilus influenzae, yet there are few data on their antibody responses to the H. influenzae type b vaccines. METHODS. We evaluated antibody responses in 248 men who were randomly assigned to receive a single dose of either the H. influenzae type b polysaccharide (PRP) vaccine or the polysaccharide-mutant diphtheria toxoid conjugate vaccine (PRP-CRM). The subjects were stratified into four groups: seronegative men (67 subjects), men with asymptomatic HIV infection (79), men with symptomatic HIV infection (47), and men with the acquired immunodeficiency syndrome (AIDS) (55). RESULTS. Before immunization, the subjects with AIDS had the lowest PRP-antibody titers; 40 percent had titers below the putative protective level (less than 0.15 micrograms per milliliter). In the seronegative subjects, those with asymptomatic HIV infection, and those with symptomatic HIV infection, the PRP-CRM vaccine led to a threefold greater increase in geometric mean antibody titers than did the PRP vaccine (P less than 0.01). However, the subjects with AIDS had a greater antibody response to the PRP vaccine. The antibody response of HIV-seropositive men to the PRP-CRM vaccine correlated significantly with the number of CD4 lymphocytes (r = 0.47, P less than 0.0001, as compared with r = -0.01 for the PRP vaccine). In these HIV-infected men, both vaccines elicited the dominant anti-PRP idiotype described previously in populations not infected with HIV. CONCLUSIONS. Immunization with the PRP-CRM conjugate vaccine early in the course of HIV infection is likely to confer protection against disease caused by H. influenzae type b.     

Monoclonal antibody solution hybridization assay for detection of human immunodeficiency virus nucleic acids.

In this report we describe a novel, nonisotopic hybridization assay for the measurement of viral RNA in biological samples. The assay involved a solution-phase reaction between a biotinylated DNA probe and RNA target sequences. Labeled hybrids were detected in an immunoreaction by using a solid-phase anti-biotin antibody and an enzyme-labeled monoclonal antibody specific for DNA-RNA hybrids. This monoclonal antibody solution hybridization assay was compared with an antigen-capture immunoassay for the detection of human immunodeficiency virus in 436 cell culture samples from 60 seropositive patients. The sensitivity and specificity of the hybridization assay were 93.5 and 94.6%, respectively. Detection of human immunodeficiency virus solely by hybridization in the initial sample but not subsequent samples from seven cultures may reflect detection of virus that was present in the patients' lymphocytes but did not replicate in vitro. Since the assay method is adapatable to the detection of either RNA or DNA, it could provide a means for the detection of a wide range of viral nucleic acids.