Peter L. Nara

MAP 30: a new inhibitor of HIV-1 infection and replication.

A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordica charantia. This compound, MAP 30 (Momordica Anti‐HIV Protein), is a basic protein of about 30 kDa. It exhibits dose‐dependent inhibition of cell‐free HIV‐1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM‐ss monolayers; (II) viral core protein p24 expression; and (iii) viral‐associated reverse transcriptase (RT) activity in HIV‐1 infected H9 cells. The doses required for 50% inhibition (ID50) in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV‐1 infections. The sequence of the N‐terminal 44 amino acids of MAP 30 has been determined.

Neutralization of HIV-1: a paradox of humoral proportions.

The production of immunoglobulin capable of neutralizing the infectivity of a virus represents one of the most remarkable molecular accomplishments of the hosts available immune defenses. It should be no surprise that a virus that has existed in the parenchyma of the immune system has evolved as an equally dynamic molecule (i.e., viral envelope) for survival. Neutralizing immunoglobulin (Ig) can best serve the host under conditions where the invading pathogen requires a well‐defined cell‐free state for establishing an infection or transmission. Evidence for a controlling and therefore protective role of neutralizing Ig against lentiviruses has been defined in natural and experimental infections with equine infectious anemia virus of ungulate members in the family equidae. Rapid replication of the virus immediately after infection and its release in a cell‐free state leads to the production of neutralizing Ig and subsequent control of the primary viremia. A similar cause‐effect relationship exists in humans between the high‐titered viremia, observed shortly after HIV‐1 infection, and the subsequent production of neutralizing Ig. Partially controlling this acute stage of viral replication by neutralizing Ig and thus preventing an otherwise acute form of immunosuppression or immune complex disease may be viewed paradoxically as a survival property of the virus. Immunologically mediated control, in a Darwinian sense, selects for viruses that have optimized the parameters of longevity and transmission from host to host. This paradox of neutralization in HIV‐1 infection appears to be mediated by the convergence of structural and functional roles of the third variable domain (V3) of the external envelope glycoprotein. During infection or envelope‐based vaccination, antibody to this cross‐reactive, immunodominant epitope dominates the antigenic repertoire. Once this occurs, the host is less able to respond to emerging viruses containing closely related V3 structures. Thus a relatively restricted clonal‐dominance of the neutralization response results. The V3 domain, apparently in concert with the rest of the molecule, provides an epitope that can tolerate and utilize its conformational flexibility to allow immune escape while maintaining its functional role in infectivity. Sixteen other putative epitopes have been described as being involved in the induction of neutralizing Ig. Currently the biologically functional role of neutralizing Ig to these other epitopes are complicated by a prior lack of knowledge and appreciation of the in vitro variables affecting their measurements. Thus it appears that the mechanisms of neutralization, the immunologic complexities of the viral envelope, and the design of in vitro assays to more accurately reflect in vivo conditions should greatly improve our future chances of designing, identifying, and eliciting protective immune responses through vaccination.—Nara, P. L.; Garrity, R. R.; Goudsmit, J. Neutralization of HIV‐1: a paradox of humoral proportions. FASEB J. 5: 2437–2455; 1991.

Qualitative and quantitative detection of HIV-1 RNA by nucleic acid sequence-based amplification.

AimTo develop a method to detect HIV-1 viral RNA by amplifying a specific nucleic acid sequence. MethodThe nucleic acid sequence-based amplification (NASBA) method uses the simultaneous activity of avian myeloblastosis virus reverse transcriptase, T7 RNA polymerase and RNase H to amplify a specific nucleic acid target sequence. ValidationAn in vitro cultured HIV-1 stock solution was used to validate the NASBA method and determine the variation in RNA measurement. ConclusionAlthough NASBA is theoretically capable of specific amplification of RNA or DNA, it is most suitable for amplification of RNA, and therefore for detection of HIV-1 viral RNA.

Genomic diversity and antigenic variation of HIV-1: links between pathogenesis, epidemiology and vaccine development.

Recent analysis of primate lentivirus genomes (1) indicates that lentiviruses have infected primates for hundreds of years. The pathogenicity of such viruses may fluctuate due to the high evolution rate of some parts of the viral genome. Fixed nucleic acid substitutions in the gag gene appear to be caused by random fixation of selectively neutral mutants, whereas nonrandom fixation of selectively advantageous mutants, as has been observed for MHC molecules and serine protease inhibitors, appears to be operational for some hypervariable env gene regions. The former is characterized by an excess of silent mutations independent of the rate of change, the latter by an excess of nonsilent mutations. This latter type of selection may especially characterize the third variable region of the external HIV envelope (V3), which contains the principal neutralization domain.—Goudsmit, J.; Back, N. K. T.; Nara, P. L. Genomic diversity and antigenic variation of HIV‐1: links between pathogenesis, epidemiology, and vaccine development. FASEB J. 5: 2427–2436; 1991.

HIV-specific immunity following immunization with HIV synthetic envelope peptides in asymptomatic HIV-infected patients.

OBJECTIVE A phase I trial was conducted to evaluate the safety and immunogenicity of an HIV synthetic peptide vaccine in HIV-seropositive individuals. The immunogens used in this study were PCLUS 3-18MN and PCLUS 6.1-18MN envelope peptides. METHODS Eight HIV-infected patients received six subcutaneous injections of 160 microg PCLUS 3-18MN in Montanide ISA 51 and were followed longitudinally for a year after the first immunization. Peripheral blood mononuclear cells (PBMC) were tested for peptide-specific T helper and cytotoxic T cell (CTL) responses, HIV-1MN neutralizing antibodies and antibodies against HIV PCLUS 3 and P18 MN peptides. RESULTS PCLUS 3-1 8MN-specific T helper responses were significantly increased at 36 weeks (P < 0.05, after adjustment for multiple comparisons) following initial immunization with PCLUS 3-18MN. A P18MN-specific CTL response, not present prior to vaccination, was observed after immunization in one patient. Serum HIV-1 MN-neutralizing antibody titers increased in each of the three patients who had low titers prior to immunization. Plasma HIV RNA levels and CD4 cell counts did not change appreciably during the study period. CONCLUSIONS This trial demonstrates that both peptides can be safely administered to HIV-infected individuals and that PCLUS 3-18MN induces increases in HIV peptide-specific immune responses.

What role do periodontal pathogens play in osteoarthritis and periprosthetic joint infections of the knee

Through the use of polymerase chain reaction (PCR)-electron spray ionization (ESI)-time of flight (TOF)-mass spectrometry (MS), we identified multiple periodontal pathogens within joint tissues of individuals undergoing replacement arthroplasties of the knee. The most prevalent of the periodontal pathogens were Treponema denticola and Enterococcus faecalis, the latter of which is commonly associated with apical periodontitis. These findings were unique to periprosthetic joint infections (PJI) of the knee and were never observed for PJIs of other lower extremity joints (hip and ankle) or upper extremity joints (shoulder and elbow). These data were confirmed by multiple independent methodologies including fluorescent in situ hybridization (FISH) which showed the bacteria deeply penetrated inside the diseased tissues, and 454-based deep 16S rDNA sequencing. The site-specificity, the tissue investment, and the identical findings by multiple nucleic-acid-based techniques strongly suggests the presence of infecting bacteria within these diseased anatomic sites. Subsequently, as part of a control program using PCR-ESI-TOF-MS, we again detected these same periodontal pathogens in aspirates from patients with osteoarthritis who were undergoing primary arthroplasty of the knee and thus who had no history of orthopedic implants. This latter finding raises the question of whether hematogenic spread of periodontal pathogens to the knee play a primary or secondary-exacerbatory role in osteoarthritis.

A cytopathic infectivity assay of human immunodeficiency virus type 1 in human primary macrophages

In addition to CD4+ T lymphocytes, cells of monocyte/macrophage lineage are a major target for human immunodeficiency virus type 1 (HIV-1) infection. In vitro studies of HIV-1 infection in human monocyte-derived macrophages can be undertaken by a reproducible cell-based assay. A macrophage-based infectivity assay was developed based on the semi-quantitative scoring of HIV-1 induced cytopathology in monolayer macrophage cultures. The assay exhibited dilution-dependent linearity with all three primary macrophage-tropic isolates tested. The end-point infectivity titers determined by this assay correlated with the results obtained by detecting viral p24 antigen in the culture supernatant. The applications of the assay in both neutralization and anti-viral protocols yielded identical results with the more time-consuming and costly p24 formats. Since the assay offers a simple and low-cost method of measuring HIV-1 infectivity in human primary macrophages, it can be used quite easily for large-scale screening or evaluation of candidate vaccines and anti-viral agents.

Plant Proteins with Antiviral Activity Against Human Immunodeficiency Virus

Human Immunodeficiency Virus (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), is a member of the lentiviruses, a subfamily of retroviruses. Unlike other retro-oncoviruses, HIV is not known to cause cancer in humans and other animals, but it does present a formidable challenge to the host. HIV integrates its genetic information into the genome of the host. The viral genome contains many regulatory elements that allow the virus to control its rate of replication in both resting and dividing cells. Most importantly, HIV infects and invades cells of the immune system and renders the patient susceptible to opportunistic infections and neoplasms.