Genetic analysis of viral variants selected in transmission of human immunodeficiency viruses to newborns.

Our previous studies have indicated that HIV transmission from infected mothers to infants occurs with viruses showing rapid kinetics of replication, and either resistance to maternal neutralizing antibodies or sensitivity to enhancing antibodies. The genotypic patterns that result in these and other phenotypic viral characteristics may provide clues to the selection pressures exerted during this mode of transmission. For this reason, DNA sequences of the envelope gene (env) were determined for viral isolates obtained from seropositive women who were mothers of either infected or uninfected infants. Sequences of viruses isolated early in life from the infected newborns were also determined, such that diversity both within isolates and between maternal and infant isolates could be assessed. Among isolates obtained from mothers of uninfected infants, the V3 region of env demonstrated a higher degree of heterogeneity than those from mothers of infected infants. Similar to the viruses obtained from the mothers of infected infants, the infant-derived viral sequences were relatively homogeneous. Finally, the reactivity of maternal plasma with infant-derived HIV isolates, whether via neutralizing or enhancing antibodies, appeared to predict the distribution of viral sequences in the infant isolates. These data suggest that selective pressure on HIV-1 during transmission or growth in the infected infant may be mediated by biologic and/or immunologic processes.

A human monoclonal antibody to HIV-1 gp41 with neutralizing activity against diverse laboratory isolates.

A potential component that may be useful for passive immunotherapy for HIV-1 is human monoclonal antibodies (HumAbs) possessing potent anti-HIV-1 activity that is directed against conserved regions of the envelope glycoprotein. Such antibodies would, in principle, have the ability to neutralize diverse isolates of HIV-1. To develop such reagents, hybridomas were derived by initial Epstein Barr virus transformation of peripheral blood mononuclear cells (PBMCs) from an asymptomatic HIV-1 seropositive donor followed by fusion with heteromyelomas, and secreted anti-HIV-1 antibodies were further characterized. The specificity of one HumAb, designated as clone 3, was determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses that indicated reactivity to the transmembrane envelope glyco-protein gp41. Synthetic pentadecapeptides overlapping by 10 amino acids were utilized for epitope mapping of clone 3; a decapeptide GCSGKLICTT in the transmembrane gp41 was identified as the epitope. Clone 3 bound to SupT1 cells infected with HTLV-IIIB in fluorescent activated cell sorting analysis. In addition, in vitro biological assays demonstrated that clone 3 possessed neutralization reactivity against diverse laboratory isolates as well as an AZT-resistant isolate. Therefore, clone 3 reactivity defines a conserved neutralizable site on the HIV-1 transmembrane glycoprotein. Clone 3 and the conserved immunogenic epitope on gp41 could be useful in passive and active immunotherapy for the acquired immunodeficiency syndrome (AIDS).

Features of HIV-1 that could influence maternal-child transmission.

OBJECTIVE: To evaluate the biological and serological properties of the human immunodeficiency virus type 1 (HIV-1) for factors potentially involved in the mother-to-child transmission of HIV-1. DESIGN: Isolates of HIV-1 were recovered from the blood of 12 of 44 nontransmitting mothers and six of eight transmitting mothers and their corresponding infants. These 24 HIV-1 isolates were compared for their biological and immunologic properties to discern any parameters that correlate with vertical transmission of HIV-1. MAIN OUTCOME MEASURES: Replication capabilities of the above-mentioned HIV-1 isolates in human peripheral blood mononuclear cells (PBMCs), human macrophages, and various T-cell lines and the susceptibilities of the viruses to neutralization or enhancement by anti-HIV-1 antibodies in autologous serum samples from mothers and infants. SETTING: San Francisco Bay Area, California. PARTICIPANTS: A cohort of 52 HIV-1-infected women and their infants in a prospective study on perinatal HIV transmission by the Bay Area Perinatal AIDS Center. RESULTS: The viral isolates from the transmitting mothers and their infants differed from the isolates from the nontransmitting mothers in their efficient replication in human PBMCs and in their ability to infect one or more human T-lymphocytic cell lines. All the HIV-1 isolates were able to infect human macrophages with only low-level replication and were unable to form syncytia in the MT-2-lymphocytic cells. No correlation between transmission and reactivity of maternal serum samples to the peptide corresponding to the principal neutralization domain of the third hypervariable region of the viral envelope was observed. However, the majority (9/12) of maternal isolates from the nontransmitters were neutralized by their autologous serum samples compared with only two among six in the transmitter group (P < .07). Moreover, five infant isolates were resistant to neutralization by their respective mother's serum samples, and one was sensitive to infection enhancement by the mother's serum. Another infant isolate was enhanced by his autologous serum. CONCLUSIONS: Viral factors that appeared to correlate with mother-to-child transmission of HIV-1 observed in a small cohort included rapid or high-titered replication in human PBMCs, T-cell line tropism, and resistance to neutralization or a sensitivity to enhancement of infection by the maternal serum.

Biological, serological, and genetic characterization of HIV-1 subtype E isolates from northern Thailand.

Twenty-three HIV-1 isolates were recovered from PBMCs from 26 HIV-1-seropositive individuals in northern Thailand. The viruses grew readily in human PBMCs but only 7 of 17 (41.2%) and 5 of 17 (29.4%) replicated and only at a low level in primary macrophages and in established T cell lines, respectively. By immunoblot assays, sera from Thai subjects were strongly reactive with gp120 from a Thailand isolate, moderately reactive with a Rwandan isolate, and weakly reactive with a North American strain. These three viruses represent, respectively, examples of subtypes E, A, and B as classified by the sequences of the envelope region. Serological assays indicated that broadly reactive rather than type-specific neutralizing activity was detected among these northern Thai sera. The majority of the sera (approximately 75%) neutralized a representative Thailand isolate and the Rwanda isolate but only 55% neutralized the North American strain. However, the difference was not statistically significant. The genetic analyses indicated that nearly all the Thai isolates were highly homogeneous and distinct from the North American/European consensus sequence (subtype B); they belong to subtype E. This is the first report providing biological, serological, and genetic characterization of HIV-1 strains from Thailand. The findings suggest these viruses were recently introduced into the country and that serological evaluation of viral strains needs to be considered along with genetic subtyping when developing an HIV-1 vaccine.

Characterization of human immunodeficiency virus type 1 isolates from children in Romania: identification of a new envelope subtype.

Human immunodeficiency virus type 1 (HIV-1) isolates recovered from infected children in Romania were characterized for their biologic, serologic, and molecular properties. The isolates were from subjects in different clinical states, and all showed cytopathic properties in peripheral blood mononuclear cells and varying kinetics of replication. The isolates grew to varying titers in macrophages and established T cell lines. Serologic evaluation with Romanian sera indicated stronger antibody response to the gp120 of Romanian isolates than to the envelope protein of HIV-1 isolates from other countries. Although there was cross-neutralization among the Romanian isolates, no substantial activity was noted against HIV-1 prototype strains from the United States, Africa, and Thailand. Genetic analysis of the envelope C2-V3 region strongly suggests that the Romanian isolates are a subtype distinct from those assigned to other HIV-1 strains analyzed to date. This finding raises questions about the origin of HIV-1 in Romania.

V3 variability can influence the ability of an antibody to neutralize or enhance infection by diverse strains of human immunodeficiency virus type 1.

Human monoclonal antibodies (mAbs) to two contiguous epitopes in the V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope have shown different effects on three distinct strains of the virus: neutralization, enhancement, or resistance to both processes. Only one amino acid in the mAb epitopes proximal to the crown of the V3 loop was different among these three strains. Substitution of this amino acid in the neutralizable strain with the amino acid of the neutralization-resistant strain or the enhanceable strain resulted in loss of both activities. The conversion of this single amino acid in the neutralization-resistant strain to that of the amino acid found in the neutralization-sensitive strain did not confer the ability for the virus to be neutralized. However, additional changes in neighboring amino acids in the V3 loop succeeded in conferring the neutralization capability. These observations indicate that one antibody species can exert three different effects on various HIV-1 strains. They could explain the emergence of neutralization "escape" variants in the presence of the neutralizing antibodies. Moreover, the results suggest caution in immunization of individuals with the envelope region from one strain since the antibodies induced may show a neutralizing effect against the homologous strain but enhancing effects against other unrelated strains.

Serum antibodies to HIV-1 in recombinant vaccinia virus recipients boosted with purified recombinant gp160. NIAID AIDS Vaccine Clinical Trials Network.

Serum antibody responses were studied in detail in four vaccinia-naive volunteers in a phase I trial evaluating primary vaccination with a recombinant vaccinia virus expressing the HIV-1 gp160 envelope glycoprotein (HIVAC-1e, Oncogen/Bristol-Myers Squibb), followed by booster immunization with baculovirus-derived rgp160 (VaxSyn, MicroGeneSys). Prior to boosting, low-titer Fc receptor (FcR)-mediated, antibody-dependent enhancing (ADE) activity was detected in two of four volunteers but no IgM, IgG, IgA, neutralizing activity, or complement-mediated ADE activity was detected. Two weeks after boosting, all four volunteers developed HIV-1-specific IgG with titers of 1:160 to 1:640 by immunofluorescence assay. IgG1 was present in sera from each individual, while IgG2 and IgG3 were present in sera from two individuals, and IgG4 was present in serum from one individual. IgM and IgA were undetectable in all sera. Only one volunteer had IgG to the heterologous HIV-1 isolates, RF, MN, and SF2, after boosting. Serum from this volunteer neutralized the vaccine strain, LAV/IIIB, but not the heterologous strains, RF, MN, and SF2. Antibodies from the remaining volunteers had no neutralizing activity. The neutralizing serum had a positive reaction in a peptide-based ELISA utilizing a peptide corresponding to the principal neutralizing domain of the third hypervariable region (i.e., V3 loop) of the envelope glycoprotein. Neutralizing activity was partially removed by adsorption to this peptide, suggesting that it contained a type-specific neutralizing vaccine epitope. A low titer (1:40 to 1:80) of complement-mediated ADE activity to HIV-1 IIIB was present in sera from three vaccinees after boosting. FcR-ADE activity for HIV-1 SF2 and SF-128A were present in sera from two of these three vaccinees. None of the volunteers developed antisyncytial antibodies. These results indicate that inoculation with recombinant vaccinia followed by rgp160 boosting is the most effective strategy to date for inducing serum antibodies to the envelope glycoproteins of HIV-1, but further study is needed to optimize the functionality and cross-reactivity of these responses.

Antibody-enhanced infection of monocytes as the pathogenetic mechanism for severe dengue illness.

Antibody-dependent enhancement of certain virus infections can occur in cells expressing Fc receptors. This mechanism plays an important pathogenetic role in the development of complications associated with dengue virus infection, including dengue hemorrhagic infection and dengue shock syndrome. The virulence of the virus, characterized by the ability to infect Fc receptor-bearing monocytes also influences the development of these severe illnesses.

Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever.

Serum specimens collected during a prospective study of dengue infections among schoolchildren in Bangkok were tested for their ability to enhance dengue 2 (DEN-2) virus growth in human monocytes in vitro. Two groups of dengue-immune sera were compared: 32 dengue antibody positive serum specimens from children who subsequently developed asymptomatic secondary dengue infections; and 9 dengue antibody positive serum specimens from children who subsequently developed severe symptomatic secondary dengue infections, 8 of which were clinically diagnosed as dengue hemorrhagic fever. Antibody-dependent enhancement of virus growth was quantitated by measurement of virus yields in supernatant fluids of normal human monocyte cultures that were infected with DEN-2 virus in the presence of undiluted test serum. Only 4 of 32 (12%) preinfection sera from asymptomatic children, but 6 of 9 (67%) preinfection sera from symptomatic children, had significant enhancing activity (P less than 0.001). High serum DEN-2 antibody dependent enhancing activity is a significant (relative risk = 6.2) risk factor for severe illness among children in a dengue hemorrhagic fever endemic region. Dengue antibodies can be neutralizing and therefore protective, or they can be enhancing and increase the risk of dengue hemorrhagic fever.

Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants.

To establish the role of maternal dengue-specific antibodies in the development of dengue hemorrhagic fever and dengue shock syndrome caused by dengue 2 virus in infants, we examined sera from mothers of infants and toddlers with dengue hemorrhagic fever or dengue shock syndrome and mothers of infants with pyrexia of unknown origin. The mean titers of hemagglutination inhibition, neutralization, and infection-enhancing activities against dengue 2 virus were not statistically different among the three groups. However, among infants who developed dengue hemorrhagic fever/dengue shock syndrome there was a strong correlation between the mothers' dengue 2 neutralizing titers and infant age at the time of onset of severe illness, where no such correlation was found among the other two groups. Furthermore, the actual age at which dengue hemorrhagic fever/dengue shock syndrome occurred in each infant correlated with the age at which maximum enhancing activity for dengue 2 infection in mononuclear phagocytes was predicted. This critical time for the occurrence of dengue hemorrhagic fever/dengue shock syndrome was observed to be approximately 2 months after the time calculated for maternal dengue 2 neutralizing antibodies to degrade below a protective level. In addition, sera of mothers of infants with dengue hemorrhagic fever/dengue shock syndrome enhanced dengue 2 virus infection to a slightly greater degree than did sera from mothers of infants with pyrexia of unknown origin and toddlers with dengue hemorrhagic fever/dengue shock syndrome. These data are consistent with the hypothesis that maternal dengue antibodies play a dual role by first protecting and later increasing the risk of development of dengue hemorrhagic fever/dengue shock syndrome in infants who become infected by dengue 2 virus.

The association of enhancing antibodies with seroconversion in humans receiving a dengue-2 live-virus vaccine.

A group of human subjects, some with yellow fever (YF) antibodies, volunteered for testing of a live-attenuated dengue-2 (DEN-2) vaccine. Serum samples taken before DEN-2 vaccination were tested for their ability to enhance infection of human monocytes by DEN-2 virus. A significantly greater proportion of enhancing antibodies (Eab) were found in YF-immune (YFI) individuals (50%) as compared to those with no evidence of flavivirus infection (9.5%). Geometric mean titers of neutralizing and hemagglutination inhibiting antibodies to DEN-2 virus in YFI subjects with Eab were fourfold to seven-fold higher than in the YFI subjects without Eab in prevaccine sera and 10- to 35-fold higher than in non-immune volunteers. Additionally, levels of Eab in prevaccine sera were directly related to antibody titers found in postvaccine sera. The presence of Eab in the serum of a human subject before DEN-2 vaccination was a good predictor of the immune response after vaccination, and may in part be responsible for the higher seroconversion rate in YF immunes (90%) as compared to nonimmunes (61%) receiving this vaccine. This is the first human study to demonstrate that circulating Eab in non-DEN-immune persons is associated with an augmented immune response to DEN virus infection. This finding supports the hypothesis that cross-reactive antibodies against one flavivirus enhance an infection with another closely related flavivirus.

Role of antibodies and host cells in plaque enhancement of Murray Valley encephalitis virus.

Chicken antisera to Murray Valley encephalitis (MVE) virus, when incubated with virus and assayed for plaques on chicken embryo (CE) monolayers, neutralized MVE virus at high concentrations of antibody, but caused increases in plaque counts at low concentrations of antibody. Plaque enhancement did not occur when the same virus-antibody mixtures were assayed on a continuous line of rhesus monkey kidney cells (LLC-MK2), nor when the anti-MVE antibody was of mammalian origin and the assay system was CE monolayers. Correspondingly, chicken anti-MVE did not enhance the plaque formation of MVE virus in a stable line of mouse macrophages, P-388D1, whereas rabbit and mouse anti-MVE did enhance plaque formation. This enhancing activity was associated with noncytophilic immunoglobulin G (IgG). The Fc terminus of the IgG molecule was required, as no plaque enhancement occurred with chicken anti-MVE Fab. These data indicate that there is a requirement for taxonomic complementarity between Fc termini and Fc receptors in the above systems. CE cell monolayers were found to contain approximately 2% of Fc receptor-bearing cells among the fibroblast-like cells. Fc receptor-bearing cells in CE monolayers were isolated and found to be of the mononuclear phagocytic lineage. These mononuclear phagocytes, which originate in lymphoid tissues and blood associated with CE tissue fragments, are integrated into primary CE monolayers and form infectious centers in the presence of virus and low dilutions of antibody.

Comparison of P388D1 mouse macrophage cell line and human monocytes for assay of dengue-2 infection-enhancing antibodies.

Tissue culture-adapted dengue 2 virus (DEN 2), strain 16681, exhibits antibody-dependent enhancement of infection (ADE) in P388D1 cells, a mouse macrophage-like cell line. ADE is dependent upon maintaining DEN 2 multiplicity of infection at between 0.1 and 0.001, and can be simply measured in multi-well plastic plates. The assay uses either trypsinized or non-trypsinized P388D1 cells at 5 x 10(5) cells per ml, an appropriate dilution of DEN 2 virus, and a source of antibody, and is most conveniently performed without further washing of stationary cultures, which are incubated in 5% CO2. Trypsinization of P388D1 cells prior to the addition of virus-serum mixtures reduced infection in control cultures thus increasing ADE. When cells were washed after incubation of virus-serum mixtures for 1 hour, a paradoxical increase of infection in cultures exposed to virus plus normal serum was noted, which reduced the sensitivity of the ADE assay. Using human cord blood sera, ADE titers measured in human monocytes and P388D1 cells were closely similar. This convenient and economical assay will facilitate large scale biological and epidemiological studies of dengue virus enhancing antibodies.