Human immunodeficiency virus type 1 (HIV-1) gp120-specific antibodies in neonates receiving an HIV-1 recombinant gp120 vaccine.

Infants born to human immunodeficiency virus type 1 (HIV-1)-infected mothers were immunized at birth and at ages 4, 12, and 20 weeks with low-, medium-, or high-dose recombinant gp120 vaccine with MF59 adjuvant (HIV-1(SF-2); n=52) or with MF59 alone as a placebo (n=9). An accelerated schedule (birth and ages 2, 8, and 20 weeks) was used for an additional 10 infants receiving the defined optimal dose and for 3 infants receiving placebo. At 24 weeks, anti-gp120 ELISA titers were greater for vaccine-immunized than for placebo-immunized infants on both schedules, and 87% of vaccinees had a vaccine-induced antibody response. At 12 weeks, antibody titers of infants on the accelerated vaccine schedule exceeded those of infants receiving placebo (4949 vs. 551; P=.01), and 63% of the vaccinees met the response criteria. Thus, an accelerated schedule of gp120 vaccinations generated an antibody response to HIV-1 envelope distinct from transplacental maternal antibody by age 12 weeks. These results provide support for further studies of vaccine strategies to prevent mother-to-infant HIV-1 transmission.

An acellular pertussis vaccine in healthy adults: safety and immunogenicity. Pennridge Pediatric Associates.

Recent data indicate that Bordetella pertussis can be an important cause of illness in adolescents and adults. In a randomized observer- and subject-blinded study, adults (> or = 18 years of age) received an acellular pertussis (aP) vaccine containing genetically inactivated pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN), or a saline placebo, and were monitored for safety and immunogenicity. IgG antibodies to PT, FHA, and PRN were measured by enzyme-linked immunosorbent assay (ELISA) and PT neutralization by a Chinese hamster ovary (CHO) cell assay. Local reactions, more common in the aP group, were mild and transient. One month after immunization, geometric mean ELISA antibody concentrations for the aP and placebo groups, respectively, were: anti-PT, 463 and 7.6; anti-FHA, 417 and 18; and anti-PRN, 855 and 14. The anti-PT neutralization titers for the aP and placebo groups were 1:3439 and 1:58 respectively. This aP vaccine is a safe and immunogenic candidate booster vaccine against pertussis for adults.

Cross-subtype neutralizing antibodies induced in baboons by a subtype E gp120 immunogen based on an R5 primary human immunodeficiency virus type 1 envelope.

Global human immunodeficiency virus type 1 (HIV-1) diversity may require engineering vaccines to express antigens representing strains prevalent in the target population of vaccine testing. The majority (90%) of incident infections in Thailand are genetic subtype E, with a small percentage of subtype B infections in the intravenous drug user populations. We have evaluated and compared the binding and HIV-1 neutralizing properties of serum antibodies induced in baboons by CHO cell-expressed monomeric gp120 derived from a CCR5-using (R5) subtype E primary HIV-1CM235 or a CXCR4-using (X4) subtype B T-cell line-adapted (TCLA) HIV-1SF2 isolate. In contrast to the subtype-specific HIV-1 neutralizing antibodies induced with recombinant HIV-1SF2 gp120 (rgp120SF2), rgp120CM235 immunization induced antibodies capable of neutralizing both subtype E and subtype B TCLA HIV-1 isolates. However, neither immunogen induced antibodies capable of neutralizing primary HIV-1 isolates. Antibody induced by rgp120CM235 preferentially bound natively folded gp120 and retained strong cross-reactivity against multiple gp120 strains within subtype E as well as subtype B. In contrast, antibody responses to rgp120SF2 were directed predominantly to linear epitopes poorly exposed on native gp120 and had more limited cross-recognition of divergent gp120. Fine epitope mapping revealed differences in antibody specificities. While both rgp120CM235 and rgp120SF2 induced antibodies to regions within C1, V1/V2, V3, and C5, unique responses were induced by rgp120CM235 to multiple epitopes within C2 and by rgp120SF2 to multiple epitopes within C3, V4, and C4. These data demonstrate that strain and/or phenotypic differences of HIV-1 subunit gp120 immunogens can substantially alter antibody binding specificities and subsequent HIV-1 neutralizing capacity.

Vaccine protection against a heterologous, non-syncytium-inducing, primary human immunodeficiency virus.

Vaccine-induced protection of chimpanzees against laboratory-adapted and syncytium-inducing, multiply passaged primary human immunodeficiency virus type 1 (HIV-1) isolates, but not against non-syncytium-inducing, minimally passaged ones, has been demonstrated. Following challenge with such an isolate, HIV-15016, we obtained complete protection in one of three chimpanzees previously protected against low- and high-dose HIV-1SF2 exposures after immunization with an adenovirus-HIV-1MN gp160 priming-HIV-1SF2 gp120 boosting regimen. At challenge, the protected chimpanzee exhibited broad humoral immunity, including neutralizing antibody activity. These results demonstrate the potential of this combination vaccine strategy and suggest that vaccine protection against an HIV isolate relevant to infection of people is feasible.

Induction of neutralizing antibodies to T-cell line-adapted and primary human immunodeficiency virus type 1 isolates with a prime-boost vaccine regimen in chimpanzees.

Five chimpanzees were immunized by administration of one or more intranasal priming doses of one to three recombinant adenoviruses containing a gp160 insert from human immunodeficiency virus type 1 (HIV-1) MN (HIV-1MN) followed by one or more boosts of recombinant HIV-1SF2 gp120 delivered intramuscularly with MF59 adjuvant. This regimen resulted in humoral immune responses in three of five animals. Humoral responses included immunochemically active anti-H1V-1 antibodies (Abs) directed to recombinant gp120 and neutralizing Abs reactive with T-cell-line-adapted HIV-1MN and HIV-1SF2. In addition, neutralizing activity was detected to the two homologous primary isolates and to two of three heterologous primary isolates which, like the immunizing strains, can use CXCR4 as a coreceptor for infection. The three animals with detectable neutralizing Abs and a fourth exhibiting the best cytotoxic T-lymphocyte response were protected from a low-dose intravenous challenge with a cell-free HIV-1SF2 primary isolate administered 4 weeks after the last boost. Animals were rested for 46 weeks and then rechallenged, without a boost, with an eightfold-higher challenge dose of HIV-1SF2. The three animals with persistent neutralizing Abs were again protected. These data show that a strong, long-lived protective Ab response can be induced with a prime-boost regimen in chimpanzees. The data suggest that in chimpanzees, the presence of neutralizing Abs correlates with protection for animals challenged intravenously with a high dose of a homologous strain of HIV-1, and they demonstrate for the first time the induction of neutralizing Abs to homologous and heterologous primary isolates.

Acellular pertussis vaccine in children with perinatal human immunodeficiency virus-type 1 infection.

The immunogenicity of an acellular pertussis vaccine containing genetically detoxified pertussis toxin, filamentous haemoagglutinin and pertactin was studied in 12 children [median age: 45 (6-107) months] with perinatal human immunodeficiency virus-type 1 (HIV-1) infection. Antibody response to all antigens was observed in six cases and another children 3 reacted to two or one antigen(s), but titres were lower than those from healthy controls. Antibody titre fold-rise correlated with preimmunization CD4-positive cell counts. Significant titres were still detectable 4 months after the third dose. The acellular vaccine is immunogenic in a portion of children with perinatal HIV-1 infection but early vaccination might be more effective, taking advantage of still adequate CD4-positive cell numbers.

Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Neutralization of a clade B primary isolate by sera from human immunodeficiency virus-uninfected recipients of candidate AIDS vaccines.

The inability of antibodies induced by experimental human immunodeficiency virus type 1 (HIV-1) vaccines to neutralize HIV-1 primary isolates may be due to a failure to elicit such antibodies, antigenic differences between the vaccine and the strains tested, insensitivity of the assays used, or to a combination of factors. New neutralization assays were used to determine the ability of candidate AIDS vaccines to generate neutralizing antibodies for clade B primary isolate BZ167, which is closely related in portions of its envelope to the immunizing strains. Sera from HIV-uninfected volunteers in vaccine trials were tested, and neutralizing activity was found in recipients of recombinant (r) gp120MN or of rgp160MN-containing canarypox boosted with rgp120SF-2. Detection of antibodies that neutralize primary isolate BZ167 correlated with neutralizing activity for homologous vaccine strains. These data demonstrate that certain candidate AIDS vaccines can elicit antibodies that neutralize a primary isolate of HIV-1.

Longitudinal analysis of quantitative virologic measures in human immunodeficiency virus-infected subjects with > or = 400 CD4 lymphocytes: implications for applying measurements to individual patients. National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group.

The natural variability of quantitative virologic measures among human immunodeficiency virus (HIV) type 1-infected persons was prospectively studied in 29 untreated persons with >600 CD4 cells/microL and in 15 persons receiving zidovudine monotherapy who had 400-550 CD4 cells/microL at study entry. Cell- and plasma-associated infectious HIV-1, provirus, and virion RNA were determined monthly as were numbers of CD4 and CD8 cells. HIV-1 replication varied widely among subjects with similar CD4 cell counts. The within-individual variability was significantly less than the variability between subjects for all virologic measures. Plasma virion HIV-1 RNA levels had the least variability. A mathematical model was devised to assess whether a potential therapeutic intervention significantly alters peripheral HIV-1 load. The model indicated that three measurements of plasma RNA would be outside the 95th percentile for the expected change in an individual due to natural variability. This approach can be used to accurately assess a therapeutic intervention among persons with low plasma HIV-1 titers.

Safety and immunogenicity of a candidate HIV-1 vaccine in healthy adults: recombinant glycoprotein (rgp) 120. A randomized, double-blind trial. NIAID AIDS Vaccine Evaluation Group.

OBJECTIVE: To evaluate the safety and immunogenicity of recombinant glycoprotein (rgp) 120, a candidate vaccine for the human immunodeficiency virus (HIV), formulated with a novel adjuvant, MF59, with or without a biological response modifier, MTP-PE. DESIGN: Multicenter, double-blind, randomized trial. SETTING: University medical centers. PARTICIPANTS: 49 healthy, HIV-seronegative volunteers 18 to 60 years of age who were at low risk for HIV type 1 (HIV-1) infection. INTERVENTIONS: In part A of the study, 32 participants were randomly assigned to receive either 15 micrograms of rgp 120 in MF59, 15 micrograms of rgp 120 in MF59 plus 50 micrograms of MTP-PE, 50 micrograms of rgp 120 in MF59, or 50 micrograms of rgp 120 in MF59 plus 50 micrograms of MTP-PE. Participants were vaccinated at 0, 1, 6, and 12 to 18 months. In part B, 17 participants were randomly assigned to receive five monthly injections of either 50 micrograms of rgp 120 in MF59 or MF59 alone followed by a booster injection at 12 to 18 months. MAIN OUTCOME MEASURES: Local and systemic reactions; laboratory measures of hepatic, renal, immunologic, and bone marrow toxicity; and HIV-specific serologic and cell-mediated immune responses. RESULTS: 13 patients in part A received 50-micrograms doses of rgp 120; type-specific neutralizing antibody responses against the SF-2 strain of HIV-1 (HIV-1/SF-2) were induced in all 13. Nine of the 13 had crossreactive neutralizing activity against the MN strain of HIV-1 (HIV-1/MN), and 2 had crossreactive neutralizing activity against the IIIB strain of HIV-1 (HIV-1/IIIB). Twelve patients had typespecific fusion inhibition activity; only 1 had crossreactive fusion inhibition activity against HIV-1/MN. The monthly vaccination schedule used in part B resulted in decreased antibody titers, indicating that a rest period in the schedule is necessary for maximal immunogenicity. Lymphoproliferative responses against gp120 were induced in all vaccine recipients. The stimulation index to gp120 was persistently greater than 15 for 6 months after the last booster vaccination was given. CD8+ cytotoxic T-lymphocyte activity was detected in 1 of the 11 participants tested. Vaccine that contained MTP-PE caused a greater number of moderate or severe local and systemic reactions (of 16 participants, 4 had local reactions and 13 had systemic reactions) than did vaccine formulated with MF59 alone (of 16 participants, 7 had local reactions [P < 0.01] and 0 had systemic reactions [P < 0.001]). CONCLUSIONS: The SF-2 rgp120 vaccine is safe and immunogenic. Three vaccinations with rgp120 in MF59 can induce type-specific and crossreactive neutralizing antibody against B-subtype laboratory strains of HIV-1. Human immunodeficiency virus-specific lymphoproliferative responses were induced in all vaccinated participants, and CD8+ cytotoxic T-lymphocyte activity was shown in one participant. A trend toward the augmentation of lymphoproliferative and humoral responses by MTP-PE was seen in the participants receiving 15 micrograms of rgp120. However, MTP-PE caused a statistically significant increase in the incidence of local and systemic side effects, which was felt to outweigh the small increase in immunogenicity provided by this biological response modifier in an otherwise well-tolerated vaccine.

Clinical, immunologic, and virologic observations related to human immunodeficiency virus (HIV) type 1 infection in a volunteer in an HIV-1 vaccine clinical trial.

A vaccine breakthrough occurred in a phase 1 clinical trial of a human immunodeficiency virus (HIV) type 1 candidate subunit vaccine. The vaccine antigen, gp120SF2, is a fully glycosylated protein produced in mammalian cells from the HIVSF2 isolate. After 4 immunizations, the subject developed neutralizing antibodies and lymphoproliferative responses to the gp120 protein. About 18 weeks after the last immunization, the subject became HIV infected. During the acute phase of infection, there was high virus burden, a decline in CD4+ T lymphocytes, increases in rgp120SF2-binding antibodies and HIVSF2- and HIVMN-neutralizing antibodies, and transient lymphoproliferative responses to HIV-1 envelope and core proteins. The nucleotide sequence of the V3 loop from 2 virus isolations displayed close similarity to the V3 sequence of the vaccine antigen. Thus, the immunologic responses induced by the vaccine in this subject did not protect him from HIV-1 infection.

Clinical and immunologic responses to human immunodeficiency virus (HIV) type 1SF2 gp120 subunit vaccine combined with MF59 adjuvant with or without muramyl tripeptide dipalmitoyl phosphatidylethanolamine in non-HIV-infected human volunteers.

A phase 1 study of 42 non-human immunodeficiency virus type 1 (HIV)-infected volunteers was initiated to determine the safety and immunogenicity of an HIV subunit vaccine consisting of recombinant envelope gp120 derived from HIVSF2 (rgp120SF2) combined with a novel adjuvant, MF59, with or without the immunomodulator muramyl tripeptide dipalmitoyl phosphatidylethanolamine (MTP-PE). All injections contained adjuvant MF59, and subjects were grouped according to MTP-PE dose. Injections were given on days 0, 30, 180, and 365. The vaccine was well tolerated with limited local and systemic reactions. These immunizations induced rgp120SF2-specific binding antibodies that persisted > or = 24 weeks. After three immunizations, all subjects receiving the antigen developed neutralizing antibodies to HIVSF2, and serum from 67% of these subjects also cross-neutralized HIVMN. ELISA-reactive antibodies to the HIVSF2 V3 region and strong lymphoproliferative responses to HIVSF2 envelope proteins were detected in all rgp120SF2-immunized subjects.

Interaction of a noncytopathic human immunodeficiency virus type 1 (HIV-1) with target cells: efficient virus entry followed by delayed expression of its RNA and protein.

Recently described noncytopathic human immunodeficiency viruses type-1 (HIV-1) form a new category within the HIV-1 family due to their unique biological properties and predominant occurrence in symptom-free individuals. To study the mechanism of noncytopathic HIV-1 infection, we compared the infectivity and life cycles of two closely related HIV-1 clones with noncytopathic (N1T-E) or cytopathic (N1T-A) properties. N1T-E virus exhibited slow kinetics of infection in T cells and monocytes. Slow infection was not due to defective virus entry, because N1T-E and N1T-A exhibited equally efficient virus-cell fusion activity and nucleocapsid internalization. Kinetic studies of N1T-E genome expression revealed low levels of viral RNA, structural proteins, and Tat protein during the first 7 days after virus entry. In contrast, cells infected with the same dose of cytopathic N1T-A virus began to express high levels of genomic RNA, structural proteins, and Tat protein within 48 hr of infection; the expression peaked on Day 5, followed by complete cell lysis. No delay in N1T-E replication, as compared to N1T-A, was observed after transfection of cloned N1T-E proviral DNA. N1T-E virus had intact Tat, Rev, and fusion functions and replicated well in chronically infected cells. These results suggest that delayed processing or expression of HIV-1 genome during the early phase of the virus replicative cycle is an important determinant in noncytopathic infection.

Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching.

Human immunodeficiency virus (HIV) was purified by sucrose gradient centrifugation and labeled with octadecylrhodamine B-chloride (R-18) under conditions resulting in 90% quenching of the fluorescence label. Incubation of R-18-labeled HIV (R-18/HIV) with CD4-positive CEM and HUT-102 cells, but not with CD4-negative MLA-144 cells, resulted in fluorescence dequenching (DQ, increase in fluorescence) of 20-25%. Similar level of DQ was observed upon incubation of CEM cells with R-18-labeled Sendai virus. DQ was observed when R-18/HIV was incubated with CD4+ cells at 37 degrees C, but not at 4 degrees C. Most of the increase in fluorescence occurred within 5 min of incubation at 37 degrees C and was independent of medium pH over the range of pH 5-8. Preincubation of cells with the lysosomotropic agent NH4Cl had no inhibitory effect on DQ. Complete inhibition was observed when target cells were fixed with glutaraldehyde prior to R-18/HIV addition. Our results demonstrate application of membrane fluorescence dequenching method to a quantitative measurement of fusion between HIV and target cell membranes. As determined by DQ, HIV penetrates into target cells by a rapid, pH-independent, receptor-mediated and specific process of fusion between viral envelope and cell plasma membrane, quite similar to that observed with Sendai virus.

Effects of differentiation inducing chemicals on in vivo malignancy and NK susceptibility of metastatic lymphoma cells.

In the present investigation we have determined the effects of the differentiation-inducing chemicals dimethyl sulfoxide (DMSO) and sodium butyrate on the growth and tumorigenicity of a highly malignant/metastatic cell line (RAW117-H10) and its less tumorigenic parental lymphoma cell line (RAW117-P). Both of these agents at doses shown to be nontoxic slowed the growth of these cells in suspension culture and significantly lengthened the doubling time while reducing colony formation in the agar tumor stem cell assay. Corresponding to these observations, the in vivo tumorigenicity of the highly malignant RAW117-H10 line was reduced by both chemicals but particularly by butyrate treatment. In addition, both agents increased the expression of some glycoproteins and the glycolipid asialo GM1. There was also a corresponding increase in the NK susceptibility of the normally NK resistant RAW117-H10 cells. To determine if the decreased malignancy of the highly malignant cells following treatment with the chemical agents was primarily due to the alteration in cell surface glycoconjugates or merely due to concomitant decreased growth potential, we transplanted highly glycosylated membrane fragments from normal syngeneic thymocytes to both RAW117-P and RAW117-H10 cells using a Sendai virus mediated membrane fusion technique. The in vivo tumorigenicity of the membrane altered RAW117-H10 cells was significantly decreased. These results strongly suggest that the decreased in vivo malignancy of RAW117-H10 cells resulting from treatment with chemical differentiation agents is caused by their increased susceptibility to NK cell mediated lysis which in turn results from cell surface changes involving altered, primarily increased, expression of certain glycosylated surface molecules. The cell surface glycoconjugates, such as receptors for certain lectins and glycolipid asialo GM1, can be used as markers for malignant potential and NK sensitivity of malignant lymphoid cells.

The detection of human minor alloantigens following restriction determinant implantation.

The recognition of minor alloantigens by cytotoxic T lymphocytes (CTL) serves as a model for the recognition of tumor and viral antigens. Progress in this area has been limited, however, since CTL recognize minor alloantigens only in association with self-class I antigens. Thus, experiments designed to study minor alloantigens are limited to target cells that share HLA determinants with the CTL. We raised CTL lines that recognized human minor alloantigens. In order to circumvent the problem that only target cells which expressed the appropriate restriction determinants could be tested for minor antigens. Sendai virus mediated fusion was used to integrate appropriate HLA antigens into cells that did not express them naturally. The target cells were then tested in CML for their expression of minor antigens. The results of experiments demonstrated that, following class I implantation, the detection of minor antigens on certain restriction determinant negative cells was possible. Furthermore, the restriction determinant was able to associate with the minor antigen in a manner appropriate for recognition by the T-cell receptor.

Non-Hodgkin's lymphoma after treatment of Hodgkin's disease: association with Epstein-Barr virus.

Non-Hodgkin's lymphoma occurs infrequently as a late complication of obscure cause after treatment of Hodgkin's disease. We investigated the possible role of Epstein-Barr virus in the pathogenesis of such secondary malignancies of B-cell lineage. Two patients, aged 25 and 43 years, developed high-grade non-Hodgkin's lymphomas 12 and 8 years after radiation therapy for Hodgkin's disease. Serologic profiles in these patients showed evidence of acute and past Epstein-Barr virus infections, respectively. Molecular hybridization analysis showed the presence of multiple cellular equivalents of virus genome in tumor specimens from each patient. Our findings suggest that Epstein-Barr virus may play an integral role in the pathogenesis of non-Hodgkin's lymphoma of B-cell lineage that develops after treatment of Hodgkin's disease.

Testing for antibodies to AIDS-associated retrovirus (HTLV-III/LAV) by indirect fixed cell immunofluorescence: specificity, sensitivity, and applications.

Seropositivity to the AIDS-associated retrovirus, HTLV-III/LAV, has profound implications. Simple and reliable tests are needed to detect such antibodies. A rapid, sensitive indirect immunofluorescence assay (IFA) on acetone-fixed virus-producing CEM/LAV-N1 cells was adapted for detection of human antibodies to HTLV-III/LAV. Specific and nonspecific patterns of of immunofluorescent reactivity were easily distinguished, and results paralleled those obtained by Western blotting and radioimmunoprecipitation (RIP), indicating that there is no need to confirm IFA positivity. In contrast, the commercial enzyme-linked immunosorbent assay (ELISA) was less reliable. False positives occurred with sera from seven hemophiliacs that were negative on Western blots, and false-negative reactions were observed on two occasions. These involved low-titer AIDS-patients' sera that were positive on Western blots, and from one of which virus was successfully isolated. Our results emphasize the requirement for confirmatory assays when the ELISA test is used for primary screening of sera for antibodies to HTLV-III/LAV. The IFA method is especially well-suited to quantitative analysis of serum antibody levels. Our data suggest that serum antibody titers rise as disease progression occurs, ultimately falling as severe complications ensue. It is suggested that in laboratories where the demand for HTLV-III/LAV antibody testing is not excessive (1,000-2,000 sera/month), IFA could serve as the only serological assay for both screening and epidemiological purposes.

Transfection of lymphoblastoid cells using DNA-loaded reconstituted Sendai virus envelopes: expression of transfected DNA and selection of transfected cells.

The American Burkitt's lymphoma cell line Loukes was cotransfected with cloned BamHI K fragment of EBV DNA and a vector pSV2neo. Reconstituted Sendai virus envelopes (RSVE) loaded with DNA were used for efficient gene transfer. Two cell lines have been obtained following culture in the presence of geneticin sulfate (G-418). Messenger RNA from both transfected DNAs was expressed during the whole period of observation, 42 days after transfection. This method provides a relatively simple and efficient means for selection of lymphoblastoid cells expressing a transfected gene.