Louis E. Henderson

Whole inactivated SIV virion vaccines with functional envelope glycoproteins: safety, immunogenicity, and activity against intrarectal challenge

Abstract: A novel type of whole inactivated simian immunodeficiency virus (SIV) virion vaccine immunogen with functional envelope glycoproteins was evaluated, without adjuvant, in rhesus macaques. Immunogens included purified inactivated virions of SIVmac239, a designed mutant of SIVmac239 with gp120 carbohydrate attachment sites deleted (SIVmac239 g4,5), and SIVmneE11S. The vaccines were noninfectious, safe, and immunogenic, inducing antibody responses and cellular responses, including responses by CD8+ lymphocytes. Interpretation of protective efficacy following intrarectal challenge was complicated by incomplete take of the challenge in some SIV naïve controls.

Experimental determination and calculations of redox potential descriptors of compounds directed against retroviral zinc fingers: Implications for rational drug design

A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of aromatic disulfides toward the NC protein of the human immunodeficiency virus type 1 (HIV‐1), NCp7, has been characterized by HPLC separation of starting reagents from reaction products. We calculated the absolute redox potentials of these compounds in the gas phase and in aqueous solvent, using a density functional theory method and a continuum solvation model. Pulsed polarography experiments were performed and showed a direct correlation between calculated and experimentally determined redox propensities. A dependence between protein reactivity and redox potential for a specific compound was shown: Reaction with NCp7 did not take place below a threshold value of redox potential. This relationship permits the distinction between active and nonactive compounds targeted against NCp7, and provides a theoretical basis for a scale of reactivity with retroviral zinc fingers. Our results indicate that electrophilic agents with adequate thiophilicity to react with retroviral NC fingers can now be designed using known or calculated electrochemical properties. This may assist in the design of antiretroviral compounds with greater specificity for NC protein. Such electrophilic agents can be used in retrovirus inactivation with the intent of preparing a whole‐killed virus vaccine formulation that exhibits unaffected surface antigenic properties.

Application of capillary zone electrophoresis for the analysis of proteins, protein-small molecules, and protein-DNA interactions

Capillary electrophoresis with a polyacrylamide-coated capillary was used for the separation of proteins, and the study of protein-small molecule and protein-DNA interactions. The coating (10% T polyacrylamide) is of sufficient thickness and hydrophilicity to reduce protein adsorption and eliminate electroosmotic flow. Data on the effect of sample solvent constituents on the peak shape and position of protein solutes was investigated. The results show that CE is a relevant and fast technique for the study of protein-DNA and protein-drug interaction. Also, the addition of TRIS-buffer to the sample solution resulted in the CE focusing of some basic proteins, while others were not affected.

Mucosal challenge of Macaca nemestrina with simian immunodeficiency virus (SIV) following SIV nucleocapsid mutant DNA vaccination.

A simian immunodeficiency virus (SIV)(Mne) DNA clone was constructed that produces viruses containing a four amino acid deletion in the second zinc finger of the nucleocapsid (NC) domain of the Gag polyprotein. Viruses produced from this clone, although non‐infectious both in vitro and in vivo, complete a majority of the steps in a single retroviral infection cycle. Eight pig‐tailed macaques (Macaca nemestrina) were inoculated intramuscularly and subcutaneously three times over the course of 24 weeks with the NC mutant expressing DNA. These macaques, and four controls, were then challenged mucosally (intrarectally) with the homologous virus (SIV Mne CL E11S) and monitored for evidence of infection and clinical disease. Prior to challenge, a measurable humoral immune response was noted in four of eight immunized macaques. After challenge, all 12 macaques became infected, although four immunized animals greatly restricted their viral replication, and one immunized animal that controlled replication remains antibody negative. No disease has been evidence during the 46‐week period of monitoring after challenge.

Reaction of HIV-1 NC p7 zinc fingers with electrophilic reagents

Publisher Summary Recently, it has been shown that Cys (X)2 Cys (X)4 His (X)4 Cys array (CCHC) zinc finger peptides are susceptible to chemical attack by a wide variety of oxidizing agents. The metal-chelated sulfur thiolates in the CCHC zinc fingers of HIV-1 p7 are known to react with a variety of chemical groups, including maleimides, nitrosos, disulfoxides, thiocarbamoyl-disulfides, and other substituted disulfides as well as oxidizing agents, such as Cu +2 , Fe +3 , and Hg +2 ions. The reaction mechanism for the thiuram disulfide class of oxidizing agents and maleimide class of alkylating agents are examined and presented in this chapter. Thiuram disulfides are examined in detail, as a member of this class of compounds, tetraethylthiuram disulfide (Antabuse) is an FDA-approved drug for alcohol abuse therapy and has very low in vivo toxicity. These compounds have functional groups that can modify zinc fingers in nucleocapsid (NC) protein and have antiviral activity but are not necessarily specific for the virus. To initiate studies leading to the design of reagents with greater specificity for the viral NC protein, it is necessary to determine the mechanism of action for model compounds and in particular to determine the initial site of attack on the NC protein. While the studies of NC protein alone demonstrated little if any cross-linking, the results with HIV-1 virus showed extensive oligomerization. The mature virion contains a compact ribonucleoprotein complex formed by the genomic RNA and ca. 2,500 copies of the NC protein. Therefore, the high concentration of NC in the viral particle the formation of intermolecular disulfide bonds over intramolecular ones is expected to be favored following virus treatment with thiuram disulfides.