David H. Schwartz

Detection and Qualification of Human Immunodeficiency Virus RNA in Patient Serum by Use of the Polymerase Chain Reaction

Human immunodeficiency virus (HIV) RNA was detected and quantified in the serum of HIV-seropositive individuals using the polymerase chain reaction (PCR) and a nonisotopic enzyme-linked affinity assay. Of 55 HIV-infected patients who were not receiving therapy, serum HIV RNA was detected in 9 of 19 who were asymptomatic, 11 of 16 with AIDS-related complex (ARC), and 18 of 20 with AIDS, with copy numbers ranging from 10(2) to greater than or equal to 5 x 10(4) 200 microliters of serum based on a relationship between absorbance and known copy number of gag gene RNA. Linear regression analysis demonstrated a correlation between infectious titer in 42 patient sera cocultured with donor peripheral blood mononuclear cells (PBMC) and PCR product absorbance (r = .70, P less than .01). Serum HIV RNA detected by PCR also correlated with serum p24 antigen positivity, CD4 counts less than 400/mm3, and the presence of HIV-related symptoms or disease. Quantification of infectious HIV RNA in cell-free serum by PCR may be useful as a marker for for disease progression or in monitoring antiviral therapy.

Immunization with Recombinant Canarypox Vectors Expressing Membrane-Anchored Glycoprotein 120 Followed by Glycoprotein 160 Boosting Fails to Generate Antibodies That Neutralize R5 Primary Isolates of Human Immunodeficiency Virus Type 1

Antibodies generated by candidate HIV-1 vaccines in a phase I clinical trial were assessed for neutralizing activity with a panel of eight well-characterized, genetically diverse clade B primary isolates having an R5 phenotype. The vaccines consisted of one of three different recombinant canarypox vectors expressing membrane-anchored HIV-1(MN)gp120 (ALVAC vCP205, vCP1433, and vCP1452) followed by boosting with a soluble gp160 hybrid consisting of MNgp120 and the majority of gp41 from strain IIIB. Serum samples from a subset of volunteers in each arm of the trial, containing moderate to high titers of neutralizing antibodies to HIV-1 MN, were analyzed. Competition assays with peptides revealed that the majority of neutralizing activity was specific for the MN-V3 loop. Despite MN-specific neutralization titers that sometimes exceeded 1:500, no neutralization of primary isolates was detected and, in some cases, mild infection enhancement was observed. In addition, little or no neutralization of the HIV-1 IIIB heterologous T cell line-adapted strain of virus was detected. These results reinforce the notion that monovalent HIV-1 ENV is a poor immunogen for generating cross-reactive neutralizing antibodies.

Induction of HIV-1-neutralising and syncytium-inhibiting antibodies in uninfected recipients of HIV-1IIIB rgp120 subunit vaccine.

A recombinant human immunodeficiency virus 1 IIIB (HIV-1IIIB) gp120 subunit vaccine (IIIB-rgp120/HIV-1, Genentech) was tested for safety and immunogenicity in a randomised, double-blind, placebo-controlled phase-I trial. HIV-1-seronegative adult volunteers received three 100 micrograms or 300 micrograms doses of IIIB-rgp120/HIV-1 in alum adjuvant (10 vaccinees in each group), or alum adjuvant alone (8 vaccinees), at 0, 4, and 32 weeks by intramuscular injection. The three injections were well tolerated in both vaccine groups. Antibodies that neutralised homologous HIV-1IIIB were induced in 9 of 10 recipients after three 300 micrograms doses, and 6 of these 9 sera also neutralised heterologous HIV-1SF2. A dose response was evident, since three 100 micrograms injections induced lower titres of HIV-1IIIB neutralising antibodies and in fewer recipients (5 of 9) than the higher dose, with no neutralisation of HIV-1SF2. Similarly, syncytia-inhibiting, CD4-rgp120-blocking, and HIV-1IIIB V3-binding antibodies were induced in a dose dependent manner. Response to the 300 micrograms per dose vaccination occurred in a larger proportion of volunteers and at higher mean titres than seen in previous human trials with other recombinant envelope subunit vaccines or live vaccinia-env priming followed by envelope subunit boosting.

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.

Safety and immunogenicity of a high-titered canarypox vaccine in combination with rgp120 in a diverse population of HIV-1-uninfected adults: AIDS vaccine evaluation group protocol 022A

&NA; To test the safety and immunogenicity of a high‐titered preparation of ALVAC‐HIV vCP205 in both high‐risk and low‐risk persons and to evaluate variations in dosing schedule, we conducted a multicenter, randomized, double‐blind trial of this vector in combination with recombinant subunit gp120 in 150 HIV‐1‐seronegative volunteers. The high‐titered ALVAC vaccine was well tolerated; adverse events were minimal and not influenced by dosing. At day 728, the cumulative probability of a cytotoxic T‐lymphocyte (CTL) response was 76% (95% confidence interval [CI]: 64%‐89%) among volunteers receiving vaccine, and the net amount attributable to vaccination was 50% (CI: 16%; 74%). The net probability of a repeated positive CTL response by day 728 was 50% (CI: 21%; 64%). There was a significant difference in CTL response at day 182 between volunteers who had received four doses versus three doses of vCP205 (42% vs. 24%, p = .052). The CTL response was similar in high‐risk volunteers and vaccinia‐naive volunteers compared with vacciniaimmune volunteers. Neutralizing antibody responses were detected in 95% of vaccinees at day 287, with higher geometric mean titers in recipients of sequential versus simultaneous dosing of the two vaccines and in vaccinia‐naive volunteers. This hightitered preparation of ALVAC‐HIV vCP205 in combination with gpl20 was safe and immunogenic in a diverse group of HIV‐1‐seronegative volunteers.

Analysis of Intercurrent Human Immunodeficiency Virus Type 1 Infections in Phase I and II Trials of Candidate AIDS Vaccines

Among 2099 uninfected subjects in phase I and II trials of candidate AIDS vaccines, 23 were diagnosed with intercurrent human immunodeficiency virus type 1 (HIV-1) infection. High-risk sexual exposures accounted for 17 infections, and intravenous drug use accounted for 6. Four subjects received placebo, 13 received a complete immunization schedule (> or = 3 injections), and 6 were partially immunized (< or = 2 injections). There was no significant difference between vaccine recipients and control groups in incidence of HIV-1 infection, virus load, CD4 lymphocyte count, or V3 loop amino acid sequence. In summary, 19 vaccinated subjects acquired HIV-1 infection during phase I and II trials, indicating that immunization with the products described is < 100% effective in preventing or rapidly clearing infection. Laboratory analysis suggested that vaccine-induced immune responses did not significantly affect the genotypic or phenotypic characteristics of transmitted virus or the early clinical course of HIV-1 infection.

Genetic and Immunologic Characterization of Viruses Infecting MN-rgp120-Vaccinated Volunteers

Proviral sequences were determined and immunologic characterization was carried out for envelope glycoproteins from 7 vaccinees who became infected with human immunodeficiency virus type 1 (HIV-1), through high-risk behavior, while participating in clinical trials of MN-rgp120, a candidate HIV-1 vaccine. All 7 infections resulted from subtype B viruses; however, only 3 of the viruses possessed the MN serotype-defining V3 domain sequence, IGPGRAF, prevalent in 60%-70% of US infections. Six of the 7 viruses differed from MN-rgp120 at a neutralizing epitope in the C4 domain, and all 7 differed from MN-rgp120 at a neutralizing epitope in the V2 domain. Recombinant gp120 was prepared from each breakthrough specimen and tested for binding to a panel of neutralizing monoclonal antibodies. The results suggest that 6 of 7 breakthrough infections may be related to incomplete immunization or to infection with viruses that differed from the vaccine immunogen at important virus-neutralizing epitopes.

Safety and effects of interleukin-2 plus zidovudine in asymptomatic individuals infected with human immunodeficiency virus.

SummaryThe safety of continuous i.v. interleukin-2 (IL-2) in conjunction with zidovudine (ZDV) was assessed in asymptomatic patients infected with human immunodeficiency virus. Clinical, immunologic, and viral parameters were monitored in a phase I/II trial with dose escalation and crossover arms. Daily doses of IL-2 from 1.5 to 12 x 106 IU/m2 were well tolerated and, in the presence of ZDV, did not induce increases in p24 antigenemia. Significant (p < 0.05) but transient increases in CD4 cells were observed midway through infusion of IL-2 at all doses, and increases in natural and lymphokine-activated killer activity were seen at higher doses. Circulating hypodense eosinophils and soluble IL-2 receptors increased more than 10-fold. Of nine patients available for long-term follow up 13–25 months from baseline and 4–21 months after stopping IL-2, six still had improved CD4 counts (versus baseline), and the mean increase (135/mm3) for all nine patients was significant (p < 0.05). Eight of these nine patients were negative for serum p24 at the start of therapy, and none had become p24 antigenemic at long-term follow-up.

V3-specific neutralizing antibodies in sera from HIV-1 gp160-immunized volunteers block virus fusion and act synergistically with human monoclonal antibody to the conformation-dependent CD4 binding site of gp120. NIH-NIAID AIDS Vaccine Clinical Trials Network.

Sera from 11 volunteers immunized with a recombinant HIV-1 gp160-expressing vaccinia virus (HIVAC-1e; Oncogen/Bristol-Myers Squibb, Seattle, WA) and boosted with baculovirus-derived rgp160 (VaxSyn; MicroGeneSys, Inc., Meriden, CT) were evaluated for functional serum antibodies and their epitopes. Sera obtained prior to boosting had undetectable HIV-1-specific IgG and neutralizing activity, and did not block HIV-1 from binding or fusing to CD4+ MT-2 cells. 14 d after boosting, sera from each volunteer contained HIV-1-specific IgG titers of 1:40 to 1:1,280. Five of these sera also contained neutralizing antibodies, where most or all neutralizing activity was blocked by a synthetic peptide corresponding to amino acids 307-330 of the V3 loop of gp120, indicating that neutralizing antibodies were mostly V3 loop-specific. All sera obtained after boosting contained HIV-1 binding/fusion-inhibition antibodies, and a significant portion of their activity was blocked by the V3 loop peptide, a result consistent with the presence of antibodies against the region of the V3 loop that participates in fusion. Three sera with V3 loop-specific neutralizing and fusion-inhibition antibodies were studied further. In competitive antibody binding experiments, antibodies reactive with the conformation-dependent, CD4 binding site of gp120 were undetectable in each serum. When evaluated in combination with a monoclonal antibody to the CD4 binding site of gp120, two sera demonstrated synergism in neutralizing assays, and all three sera demonstrated synergism in binding/fusion-inhibition assays, further indicating that the functional antibodies were primarily V3 loop-specific. The synergism also suggests that a vaccine that elicits strong serum antibody responses to both regions of gp120 may improve the potential for inducing protective immunity.

Anatomical loci of HIV-associated immune activation and association with viraemia

BACKGROUND Lymphocyte activation, associated with vaccination or infection, can be measured by positron emission tomography (PET). We investigated the ability of PET to detect and measure magnitude of lymph-node activation among asymptomatic HIV-1-infected individuals. METHODS Initially we assessed PET response in eight HIV-1-uninfected individuals who had received licensed killed influenza vaccine. In an urban teaching hospital, we recruited 12 patients recently infected with HIV-1 (<18 months since seroconversion) and 11 chronic long-term HIV-1 patients who had stable viraemia by RT-PCR (non-progressors). After injection with fluorine-18-labelled fluorodeoxyglucose, patients underwent PET. We correlated summed PET signal from nodes with viral load by linear regression on log-transformed values. FINDINGS Node activation was more localised after vaccination than after HIV-1 infection. In early and chronic HIV-1 disease, node activation was greater in cervical and axillary than in inguinal and iliac chains (p<0.0001), and summed PET signal correlated with viraemia across a 4 log range (r2=0.98, p<0.0001). Non-progressors had small numbers of persistently active nodes, most of which were surgically accessible. INTERPRETATION The anatomical restriction we noted may reflect microenvironmental niche selection, and tight correlation of PET signal with viraemia suggests target-cell activation determines steady-state viral replication.