Vera Bongertz

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

Background Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). Methods In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. Findings Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. Conclusions Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation.

Molecular epidemiology of HIV-1 in Brazil: high prevalence of HIV-1 subtype B and identification of an HIV-1 subtype D infection in the city of Rio de Janeiro Brazil.

HIV-1 genetic variability and the potential association with modes of transmission exposure categories gender and distribution over time were investigated in 1993-96 in 131 HIV-infected individuals from Rio de Janeiro Brazil. The study group included 28 homosexual and 15 bisexual men 55 heterosexuals 24 injecting drug users and 1 blood transfusion recipient. HIV-1 env subtyping by heteroduplex mobility assay identified 106 infections (80.9%) with subtype B 20 (15.3%) with subtype F and 1 (0.8%)--the first reported in Brazil--with subtype D. According to restriction fragment length polymorphism with fok 1 restriction enzyme 39 (37%) of the 106 subtype B samples had the GWGR motif at the tip of the V3 loop typically found in subtype B in Brazil. A previous study conducted in Sao Paulo Brazil suggested that subtype F could be related specifically to injecting drug use. The present study however found no significant associations between HIV-1 subtypes and exposure categories gender or mode of transmission. The long-lasting nature of the AIDS epidemic in Brazil may have favored the dispersion of the HIV-1 subtypes among the various exposure categories.

HIV-1 subtyping in Salvador, Bahia, Brazil : A city with African sociodemographic characteristics

To investigate the prevalence of the HIV-1 subtypes in different populations from Salvador, Bahia, Brazil, blood samples from 72 HIV-1-seropositive injecting drug users (IDUs) and 62 individuals infected sexually were analyzed using the heteroduplex mobility assay (HMA). In the IDU group, 89.5% were classified as subtype B, 3% as subtype F, and 7.5% showed a B/F HMA profile. In the sexual transmission (ST) group, 95% were identified as B subtype, 3.4% showed a B/F profile, and 1.6% a B/C/E HMA profile. All Brazilian samples that showed multiple reactivities in the HMA analysis clustered on sequencing with B North American/ European HIV-1 isolates in the phylogenetic analysis, whereas the F subtypes clustered with F Brazilian HIV-I isolates. Serologic reactivities of IDUs sera were examined using a panel of synthetic V3 loop peptides representative of the different HIV-1 subtypes. No difference in serologic reactivity between F and B subtype plasma could be observed. Predominance of HIV-I subtype B was identified in both study groups, whereas subtype F was detected only among IDUs in a frequency lower than described for other Brazilian regions.

The role of "long-term" and "new" injectors in a declining HIV/AIDS epidemic in Rio de Janeiro, Brazil.

Background. A substantial decline of HIV prevalence has been observed in injection drug users (IDUs) from Rio de Janeiro, in recent years. Differential characteristics and behaviors of new (injecting for <6 years) and long-term (>=6 y) injectors may help to understand recent changes and to implement appropriate preven-tion strategies. Methods. Between October 1999 and December 2001, 609 active/ex-IDUs were recruited from different communities, interviewed, and tested for HIV. Contingency table analysis and t-tests were used to assess differences between new and long-term injectors. Multiple logistic regression was used to identify independent predictors of HIV serostatus for long-term and new injectors. Results. HIV prevalence was 11.7% for 309 long-term injectors (95% CI 8.1–15.3) and 4.3% for 300 new injectors (95% CI 2.0–6.6). New injectors reported having engaged in treatment and having received syringes from needle exchange programs (NEPs) more frequently than long-term injectors in the last 6 months, but sharing behaviors remained frequent and even increased vis-à-vis long-term injectors. For male new injectors, “sexual intercourse with another man” was found to be the sole significant risk factor for HIV infection (Adj OR = 8.03; 95% CI 1.52–42.48). Among male long-term injectors, “to have ever injected with anyone infected with HIV” (Adj OR = 3.91; 95% CI 1.09–14.06) and to have “ever been in prison” (Adj OR = 2.56; 95% CI 1.05–6.24) were found to be significantly associated with HIV infection. Discussion. New injectors are seeking help in drug treatment centers or needle exchange programs. They differ from long-term injectors in terms of their risk factors for HIV infection and have lower prevalence levels for HIV. Such differences may help to understand the recent dynamics of HIV/AIDS in this population and highlight the need to reinforce new injectors’ help-seeking behavior and to reduce current unacceptably high levels of unprotected sex and syringe sharing in new injectors despite attendance of prevention/treatment programs.

Immune activation and antibody responses in non-progressing elite controller individuals infected with HIV-1

An extremely rare subset of patients infected with HIV‐1 designated as “non‐progressing elite controllers” appears to be able to maintain stable CD4+ T‐cell counts and a median plasma viremia below the detection limit of current ultrasensitive assays (<50–80 copies/ml of plasma) for >10 years in the absence of antiretroviral therapy. Lymphocyte subsets (CD4+, CD8+), immune activation markers (HLA‐DR+, CD38+, Beta‐2‐microglobulin), and HIV‐specific antibody responses were longitudinally examined in four non‐progressing elite controllers over more than 5 years. Two control groups of seronegative healthy individuals and untreated patients infected with HIV‐1 presenting detectable viremia were also included. None of the non‐progressing elite controllers displayed the high T‐cell activation levels generally seen in the seropositive individuals, keeping them within the normal range. Three non‐progressing elite controllers showed no significant immune system abnormalities when compared to seronegative individuals, displaying a low proportion of HIV‐1‐specific binding antibodies and low avidity index, similar to those observed for individuals infected recently with HIV‐1. One non‐progressing elite controller exhibited CD8+ T‐cell counts and β2‐M levels above normal ranges and developed a low but “mature” (high‐avidity) HIV‐1‐specific antibody response. Thus, the non‐progressing elite controllers are able to maintain normal T‐cell activation levels, which may contribute to prevent, or greatly reduce, the damage of the immune system typically induced by the HIV‐1 over time. They are, however, immunologically heterogeneous and very low levels of antigen exposure seem to occur in these patients, sufficient for sustaining a low, but detectable, HIV‐1‐specific immunity. J. Med. Virol. 81:1681–1690, 2009.

Is human immunodeficiency virus/acquired immunodeficiency syndrome decreasing among Brazilian injection drug users? Recent findings and how to interpret them

We briefly review findings from Brazilian settings where the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic among injection drug users (IDUs) seems to be decreasing, highlighting recent findings from Rio de Janeiro and discussing methodological alternatives. Former analyses using serologic testing algorithm for recent HIV seroconversion have shown that HIV incidence has been low in IDUs recruited by two different surveys carried out in Rio, where low injection frequencies and infection rates have been found among new injectors. The proportion of AIDS cases among IDUs in Rio has been fairly modest, compared to São Paulo and especially to the southernmost states. Notwithstanding, the interpretation of findings from serial surveys constitutes a challenge, magnified in the assessment of HIV spread among IDUs due to the dynamic nature of the drug scenes and limitations of sampling strategies targeting hard-to-reach populations. Assessment of epidemic trends may profit from the triangulation of data, but cannot avert biases associated with sampling errors. Efforts should be made to triangulate data from different sources, besides exploring specific studies from different perspectives. In an attempt to further assess the observed trends, we carried out original analyses using data from Brazilian AIDS databank.

Vertical human immunodeficiency virus type 1 - HIV-1 -transmission - a review

Several factors appear to affect vertical HIV-1 transmission, dependent mainly on characteristics of the mother (extent of immunodeficiency, co-infections, risk behaviour, nutritional status, immune response, genetical make-up), but also of the virus (phenotype, tropism) and, possibly, of the child (genetical make-up). This complex situation is compounded by the fact that the virus may have the whole gestation period, apart from variable periods between membrane rupture and birth and the breast-feeding period, to pass from the mother to the infant. It seems probable that an extensive interplay of all factors occurs, and that some factors may be more important during specific periods and other factors in other periods. Factors predominant in protection against in utero transmission may be less important for peri-natal transmission, and probably quite different from those that predominantly affect transmission by mothers milk. For instance, cytotoxic T lymphocytes will probably be unable to exert any effect during breast-feeding, while neutralizing antibodies will be unable to protect transmission by HIV transmitted through infected cells. Furthermore, some responses may be capable of controlling transmission of determined virus types, while being inadequate for controlling others. As occurrence of mixed infections and recombination of HIV-1 types is a known fact, it does not appear possible to prevent vertical HIV-1 transmission by reinforcing just one of the factors, and probably a general strategy including all known factors must be used. Recent reports have brought information on vertical HIV-1 transmission in a variety of research fields, which will have to be considered in conjunction as background for specific studies.

Immunoreactivity of brazilian HIV isolates with different V3 motifs

Viral and Rickettsial Disease Laboratory, California Department of Health Services, 2151 Berkeley Way, Berkeley, CA 94704, USA *Departamento de Immunologia, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil **Laboratorio de Immunogenetica e Transplante Experimental, Faculdade de Medicina da Universidade de Sao Paulo ***Laboratorio de Retrovirus, Servico de Virologia, Instituto Adolfo Lutz, Sao Paulo, SP, Brasil ****DIPA, Escola Paulista de Medicina, Sao Paulo, SP, Brasil *****Laboratorio Avancado de Saude Publica, Centro de Pesquisa Goncalo Moniz-FIOCRUZ, Salvador, BA, Brasil

Correlation between susceptibility of primary HIV-1 isolates to autologous and heterologous neutralizing antibodies

Objective: To study the susceptibility of primary HIV‐1 isolates towards autologous and heterologous neutralizing antibodies (NAb). Design: Blood was collected and primary HIV‐1 isolated from individuals residing in Rio de Janeiro, Brazil, in all phases of disease. Methods: Primary HIV‐1 isolates were incubated with autologous or heterologous plasma and neutralization of infection of freshly pre‐stimulated normal human peripheral blood mononuclear cells was assayed in parallel to median infectious dose determinations in the absence of antibodies. Levels of HIV‐1 p24 antigen were used for evaluation of viral neutralization. Results: Autologous neutralization (75%) was observed for 13 (52%) out of 25 of the primary HIV‐1 isolates, and 15 (71%) out of 21 isolates were susceptible to 75% heterologous neutralization by at least one‐half of the heterologous plasma tested. Primary HIV‐1 isolates susceptible to autologous NAb showed a higher susceptibility towards neutralization by heterologous NAb than isolates that could not be neutralized by the autologous plasma (P= 0.049). The susceptibility of the primary HIV‐1 isolates towards neutralization by heterologous NAb was significantly higher for isolates derived from men (P= 0.001), and for isolates obtained from individuals infected through homo‐/bisexual risk behaviour in comparison with those infected through heterosexual HIV‐1 transmission (P= 0.03). Conclusions: Susceptibility of primary HIV‐1 isolates to autologous and heterologous neutralization was significantly correlated, indicating that escape mutants may become resistant not only to autologous but also to heterologous NAb.

Trypanosoma cruzi: circulating antigens

Circulating antigens were detected in sera of mice experimentally infected with a high close of Trypanosoma cruzi by reaction with sera from chronically infected mice. The immunodiffusion reaction between homologous acute and chronic sera produced four precipitation lines. By reaction with chronic mouse serum, circulating antingens were detected in sera from heavily infected hamsters, dogs, rabbits and in sera from chagasic patients. A reaction was also found in urine from acutely infected mice and dogs. Trypanosoma cruzi exoantigen was detected in trypanosome culture medium and in the supernatant of infected cell cultures. Attempts to isolate the antigens are described.