Preliminary findings of an in vitro human spleen mononuclear cell culture system for primary isolates of HIV type 1.

Acute HIV-1 infection is often manifested with a high level of viremia. The cell types and tissues/organs that contribute to the virus load are thought to be of central and peripheral lymphoreticular origin. The establishment and permissiveness of organ-based cell culture systems from spleen with laboratory strains or primary isolates of HIV-1 have not been reported. We studied unseparated splenic mononuclear cells (SMCs) and adherent cells derived from human spleen and liver in comparison with blood monocyte-derived macrophages (MDMs). Unstimulated, SMCs were highly permissive to primary lymphotropic HIV-1 and dual/macrophage-tropic isolates (which are able to replicate in both MDMs and PBMCs). Furthermore, SMCs were found to replicate virus to high titer in a rapid log-phase manner and exhibited a prolonged stationary phase of virus production, unlike PBMCs, which required conventional activation with mitogens and exhibited a much shorter period of virus production. Interestingly, the SMCs maintained themselves as a mixed phenotype of nested lymphocytes with complex and well-differentiated macrophage(s) for extended periods of time. In addition, splenic macrophages readily purified by adherence were highly permissive to a dual/macrophage-tropic primary isolate, HIV-1ADA, intermediate with two laboratory strains, HIVR-1RF and HIV-lHXB3, and least permissive to the lymphotropic primary isolate HIV-1Mr452 and two other laboratory strains, HIV-1CC and HIV-1MN. The replication of HIV-1ADA as measured by extracellular p24 was sustained for up to 7 weeks and similar to the replication patterns observed with adherent hepatic macrophages and blood-derived MDMs. This study demonstrates that exogenous stimulation is not required for infection of these cells; either adherence-isolated and/or mixed lymphoid populations can be studied together, and viable stocks can be readily prepared and cryopreserved. In addition, these cells could be used for isolating new and/or other variants of HIV-1. Thus, the use of the SMC primary in vitro cell culture system for future studies involving HIV-1 is warranted.

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.

Human plasma enhances the infectivity of primary human immunodeficiency virus type 1 isolates in peripheral blood mononuclear cells and monocyte-derived macrophages.

Physiological microenvironments such as blood, seminal plasma, mucosal secretions, or lymphatic fluids may influence the biology of the virus-host cell and immune interactions for human immunodeficiency virus type 1 (HIV-1). Relative to media, physiological levels of human plasma were found to enhance the infectivity of HIV-1 primary isolates in both phytohemagglutinin-stimulated peripheral blood mononuclear cells and monocyte-derived macrophages. Enhancement was observed only when plasma was present during the virus-cell incubation and resulted in a 3- to 30-fold increase in virus titers in all of the four primary isolates tested. Both infectivity and virion binding experiments demonstrated a slow, time-dependent process generally requiring between 1 and 10 h. Human plasma collected in anticoagulants CPDA-1 and heparin, but not EDTA, exhibited this effect at concentrations from 90 to 40%. Furthermore, heat-inactivated plasma resulted in a loss of enhancement in peripheral blood mononuclear cells but not in monocyte-derived macrophages. Physiological concentrations of human plasma appear to recruit additional infectivity, thus increasing the infectious potential of the virus inoculum.

Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus.

To determine the factors governing inactivation and neutralization, physical, chemical, and biological assays were performed on a molecular clone of human immunodeficiency type 1 (HIV-1HXB3). This included quantitative electron microscopy, gp120 and p24 enzyme-linked immunosorbent assays, reverse, transcriptase assays, and quantitative infectivity assays. For freshly harvested stocks, the ratio of infectious to noninfectious viral particles ranged from 10(-4) to 10(-7) in viral stocks containing 10(9) to 10(10) physical particles per milliliter. There were relatively few gp120 knobs per HIV particle, mean approximately 10 when averaged over the total particle count. Each HIV particle contained a mean approximately 5 x 10(-17) g of p24 and approximately 2 x 10(-16) g of RNA polymerase, corresponding to about 1200 and 80 molecules, respectively. The spontaneous shedding of gp120 envelope proteins from virions was exponential, with a half-life approximately 30 hr. The loss of RNA polymerase activity in virons was also exponential, with a half-life approximately 40 hr. The physical breakup of virions and the dissolution of p24 core proteins were slow (half-life greater than 100 hr) compared to the gp120 shedding and polymerase loss rates. The decay of HIV-1 infectivity was found to obey superimposed single- and multihit kinetics. At short preincubation times, the loss of infectivity correlated with spontaneous shedding of gp120 from virions. At longer times, an accelerating decay rate indicated that HIV requires a minimal number of gp120 molecules for efficient infection of CD4+ cells. The blocking activity of recombinant soluble CD4 (sCD4) and phosphonoformate (foscarnet) varied with the number of gp120 molecules and number of active RNA polymerase molecules per virion, respectively. These results demonstrate that the physical state of virions greatly influences infectivity and neutralization. The knowledge gained from these findings will improve the reliability of in vitro assays, enhance the study of wild-type strains, and facilitate the evaluation of potential HIV therapeutics and vaccines.

Blocking of human immunodeficiency virus infection depends on cell density and viral stock age.

Quantitative infectivity assays were used to study how the blocking activity of soluble CD4 (sCD4) is affected by sCD4 concentration, target cell density, and viral stock age. During incubation with 20 nM sCD4, human immunodeficiency virus type 1 (HIV-1) stocks underwent irreversible inactivation. In contrast, inactivation with 2 nM sCD4 was almost entirely reversible. At lower sCD4 concentrations (less than or equal to 2 nM) and target cell densities of 6.25 x 10(4) ml-1, sCD4 blocking activity for HIV-1 gave a gp120-sCD4 association constant (Kassoc) of 1.7 x 10(9) M-1, which agrees with chemical measurements. At the higher density of 1.6 x 10(7) cells ml-1, however, the blocking activity was 20-fold less. During incubation of HIV-1 stock optimized for infectivity by rapid harvest, sCD4 blocking activity increased 20-fold during a 3-h window. These results show that competitive blocking activity depends strongly on target cell density and virion age. Thus, unappreciated variations in HIV stocks and assay conditions may hinder comparisons of blockers from laboratory to laboratory, and the age of HIV challenge stocks may influence studies of drug and vaccine efficacy. The results also suggest that blocking of viral particles in lymphoid compartments will require very high competitive blocker concentrations, which may explain the refractory outcomes from sCD4-based drug trials in humans.

HIV requires multiple gp120 molecules for CD4-mediated infection.

Binding of glycoprotein gp120 to the T cell-surface receptor CD4 is a crucial step in CD4-dependent infection of a target cell by the human immunodeficiency virus (HIV). Blocking some or all gp120 molecules on the viral surface should therefore inhibit infection. Consequently, competitive receptor inhibitors, such as soluble synthetic CD4 (sCD4), synthetic CD4 peptides and immunoglobulins, have been investigated in vitro and in vivo, but little is known about the molecular mechanisms of these inhibitors. We have now quantitatively examined blocking by soluble CD4 in the hope of gaining insight into the complex process of viral binding, adsorption and penetration. At low sCD4 concentrations, the inhibition in three HIV strains is proportional to the binding of gp120. The biological association constant (gp120-sCD4 Kassoc) for HIV-2NIHZ is (8.5 +/- 0.5) x 10(7) M-1, whereas Kassoc for HIV-1HXB3 (1.4 +/- 0.2) and HIV-1MN (1.7 +/- 0.1) x 10(9) M-1 are 15-20-fold larger. For all three viral strains, the biological Kassoc from infectivity assays is comparable to the chemical Kassoc. The inhibitory action of sCD4 at high concentrations, however, is not fully explained by simple proportionality with the binding to gp120. Positive synergy in blocking of infection occurs after about half the viral gp120s molecules are occupied, and is identical for all three viral strains, despite the large differences in Kassoc. Our method of measuring the viral-cell receptor Kassoc directly from infectivity assays is applicable to immunoglobulins, to other viruses and to assays using primary or transformed cell lines.

In vitro susceptibility of newborn murine retinal cells to herpes simplex virus type 1 infection.

We have investigated the in vitro susceptibility of murine neural retinal cells to infection by herpes simplex virus Type 1 (HSV-1). Retinal cells obtained from newborn C57B1/6 mice were cultured for 6 days and infected with varying doses of HSV-1. Infection was determined by ABC immunoperoxidase staining of fixed cultures for HSV-1 antigens. Retinal neurons, including amacrine cells, were highly susceptible to infection, with 100% of the multipolar neurons expressing viral antigens after 12 hr of infection. Glial cells and retinal pigment epithelial (RPE) cells also were 100% infected within 12-16 hr. Photoreceptor infection was not as fast, but all surviving photoreceptor cells and their precursors became infected by 24-48 hr postinoculation. Since embryonic chick photoreceptors are highly resistant to HSV-1, these results demonstrate that mammalian (murine) photoreceptor cells differ from avian photoreceptor cells in their susceptibility to in vitro HSV-1 infection. In addition, our current results suggest that the in vivo resistance of adult C57B1/6 mice to herpetic retinitis may not reside at the level of the individual retinal cell populations, although apparent differences in susceptibility exist among the various retinal cell subpopulations.

Immunogenicity versus pathogenicity after anterior chamber inoculation of an acyclovir-induced double mutant of HSV-1.

The acyclovir-induced herpes simplex virus Type 1 (HSV-1) strain, R9C2, a double mutant in thymidine kinase (TK) and DNA polymerase (DNA pol), and its parental strain SC16 were compared for their effects on ocular pathology and systemic immunity after unilateral inoculation into the anterior chamber (AC) of BALB/c mouse eyes. Although AC-injected R9C2 produced no retinal necrosis (0/18 eyes), this mutant induced active suppression (33-87%) of anti-HSV delayed type hypersensitivity similar to that induced by another HSV strain, KOS. AC-injected parental strain, SC16, caused fatal disease within 7-10 days, and induced bilateral retinal necrosis and suppression of DTH in 100% of the mice. Preimmunization with R9C2 protected mice in a dose-dependent fashion from the pathologic and lethal effects of AC-injected parental virus. These data suggest that the immunogenicity of the TK and DNA pol double mutant remains intact despite the decreased ocular and systemic pathogenicity observed after intracameral inoculation.