Indresh K. Srivastava

Atovaquone, a Broad Spectrum Antiparasitic Drug, Collapses Mitochondrial Membrane Potential in a Malarial Parasite

At present, approaches to studying mitochondrial functions in malarial parasites are quite limited because of the technical difficulties in isolating functional mitochondria in sufficient quantity and purity. We have developed a flow cytometric assay as an alternate means to study mitochondrial functions in intact erythrocytes infected with Plasmodium yoelii, a rodent malaria parasite. By using a very low concentration (2 nM) of a lipophilic cationic fluorescent probe, 3,3′dihexyloxacarbocyanine iodide, we were able to measure mitochondrial membrane potential(ΔΨm) in live intact parasitized erythrocytes through flow cytometry. The accumulation of the probe into parasite mitochondria was dependent on the presence of a membrane potential since inclusion of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, dissipated the membrane potential and abolished the probe accumulation. We tested the effect of standard mitochondrial inhibitors such as myxothiazole, antimycin, cyanide and rotenone. All of them except rotenone collapsed the ΔΨm and inhibited respiration. The assay was validated by comparing the EC50 of these compounds for inhibiting ΔΨm and respiration. This assay was used to investigate the effect of various antimalarial drugs such as chloroquine, tetracycline and a broad spectrum antiparasitic drug atovaquone. We observed that only atovaquone collapsed ΔΨm and inhibited parasite respiration within minutes after drug treatment. Furthermore, atovaquone had no effect on mammalian ΔΨm. This suggests that atovaquone, shown to inhibit mitochondrial electron transport, also depolarizes malarial mitochondria with consequent cellular damage and death.

Resistance mutations reveal the atovaquone‐binding domain of cytochrome b in malaria parasites

Atovaquone represents a class of antimicrobial agents with a broad‐spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria‐containing parasites. To that end, we derived nine independent atovaquone‐resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone‐mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance‐conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side‐chains may determine atovaquone‐binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.

Purification, Characterization, and Immunogenicity of a Soluble Trimeric Envelope Protein Containing a Partial Deletion of the V2 Loop Derived from SF162, an R5-Tropic Human Immunodeficiency Virus Type 1 Isolate

ABSTRACT The envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the major target of neutralizing antibody responses and is likely to be a critical component of an effective vaccine against AIDS. Although monomeric HIV envelope subunit vaccines (gp120) have induced high-titer antibody responses and neutralizing antibodies against laboratory-adapted HIV-1 strains, they have failed to induce neutralizing antibodies against diverse heterologous primary HIV isolates. Most probably, the reason for this failure is that the antigenic structure(s) of these previously used immunogens does not mimic that of the functional HIV envelope, which is a trimer, and thus these immunogens do not elicit high titers of relevant functional antibodies. We recently reported that an Env glycoprotein immunogen (o-gp140SF162ΔV2) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162, when used in a DNA priming-protein boosting vaccine regimen in rhesus macaques, induced neutralizing antibodies against heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIVSF162P4 virus. Here we describe the purification of this protein to homogeneity, its characterization as trimer, and its ability to induce primary isolate-neutralizing responses in rhesus macaques. Optimal mutations in the primary and secondary protease cleavage sites of the env gene were identified that resulted in the stable secretion of a trimeric Env glycoprotein in mammalian cell cultures. We determined the molecular mass and hydrodynamic radius (Rh) using a triple detector analysis (TDA) system. The molecular mass of the oligomer was found to be 324 kDa, close to the expected Mw of a HIV envelope trimer protein (330 kDa), and the hydrodynamic radius was 7.27 nm. Negative staining electron microscopy of o-gp140SF162ΔV2 showed that it is a trimer with considerable structural flexibility and supported the data obtained by TDA. The structural integrity of the purified trimeric protein was also confirmed by determinations of its ability to bind the HIV receptor, CD4, and its ability to bind a panel of well-characterized neutralizing monoclonal antibodies. No deleterious effect of V2 loop deletion was observed on the structure and conformation of the protein, and several critical neutralization epitopes were preserved and well exposed on the purified o-gp140SF162ΔV2 protein. In an intranasal priming and intramuscular boosting regimen, this protein induced high titers of functional antibodies, which neutralized the vaccine strain, i.e., SF162. These results highlight a potential role for the trimeric o-gp140SF162ΔV2 Env immunogen in a successful HIV vaccine.

Mucosal adjuvants and delivery systems for protein-, DNA- and RNA-based vaccines

Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self‐administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immunopotentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co‐administered with pathogen‐derived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administration. Although vaccines can be quite diverse in their composition, including inactivated virus, virus‐like particles and inactivated bacteria (which are inert), protein‐like vaccines, and non‐replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus‐based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines.

Changes in the immunogenic properties of soluble gp140 human immunodeficiency virus envelope constructs upon partial deletion of the second hypervariable region.

ABSTRACT Immunization of macaques with the soluble oligomeric gp140 form of the SF162 envelope (SF162gp140) or with an SF162gp140-derived construct lacking the central region of the V2 loop (ΔV2gp140) results in the generation of high titers of antibodies capable of neutralizing the homologous human immunodeficiency virus type 1 (HIV-1), SF162 virus (Barnett et al. J. Virol. 75 :5526-5540, 2001). However, the ΔV2gp140 immunogen is more effective than the SF162gp140 immunogen in eliciting the generation of antibodies capable of neutralizing heterologous HIV-1 isolates. This indicates that deletion of the V2 loop alters the immunogenicity of the SF162gp140 protein. The present studies were aimed at identifying the envelope regions whose immunogenicity is altered following V2 loop deletion. We report that the antibodies elicited by the SF162gp140 immunogen recognize elements of the V1, V2, and V3 loops, the CD4-binding site, and the C1 and C2 regions on the homologous SF162 gp120. With the exception of the V1 and V2 loops, the same regions are recognized on heterologous gp120 proteins. Surprisingly, although a minority of the SF162gp140-elicited antibodies target the V3 loop on the homologous gp120, the majority of the antibodies elicited by this immunogen that are capable of binding to the heterologous gp120s tested recognize their V3 loops. Deletion of the V2 loop has two effects. First, it alters the immunogenicity of the V3 and V1 loops, and second, it renders the C5 region immunogenic. Although deletion of the V2 loop does not result in an increase in the immunogenicity of the CD4-binding site per se, the relative ratio of anti-CD4-binding site to anti-V3 loop antibodies that bind to the heterologous gp120s tested is higher in sera collected from the ΔV2gp140-immunized animals than in the SF162gp140-immunized animals. Overall, our studies indicate that it is possible to alter the immunogenic structure of the HIV envelope by introducing specific modifications.

Purification and Characterization of Oligomeric Envelope Glycoprotein from a Primary R5 Subtype B Human Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1US4. We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (Rh ) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller Rh of the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.

Protection of macaques against vaginal SHIV challenge by systemic or mucosal and systemic vaccinations with HIV-envelope.

Background:Worldwide, the majority of human immunodeficiency virus (HIV) infections occur by heterosexual transmission. Thus, the development of a vaccine that can prevent intravaginal HIV infection is an important goal of AIDS vaccine research. Objectives:To determine which single or combination of systemic and mucosal routes of immunizations of female rhesus macaques with an HIV-1SF162 envelope protein vaccine induced protection against intravaginal challenge with SHIV. Design:Female rhesus macaques were immunized with an HIV-1SF162 envelope protein vaccine administered systemically (intramuscularly), or mucosally (intranasally), or as a sequential combination of both routes. The macaques were then challenged intravaginally with SHIVSF162P4, expressing an envelope that is closely matched (homologous) to the vaccine. Results:Macaques receiving intramuscular immunizations, alone or in combination with intranasal immunizations, were protected from infection, with no detectable plasma viral RNA, provirus, or seroconversion to nonvaccine viral proteins, and better preservation of intestinal CD4+ T cells. Serum neutralizing antibodies against the challenge virus appeared to correlate with protection. Conclusions:The results of this study demonstrate that, in the nonhuman primate model, it is possible for vaccine-elicited immune responses to prevent infection after intravaginal administration of virus.

Antibody-Mediated Protection against Mucosal Simian-Human Immunodeficiency Virus Challenge of Macaques Immunized with Alphavirus Replicon Particles and Boosted with Trimeric Envelope Glycoprotein in MF59 Adjuvant

ABSTRACT We have previously shown that rhesus macaques were partially protected against high-dose intravenous challenge with simian-human immunodeficiency virus SHIVSF162P4 following sequential immunization with alphavirus replicon particles (VRP) of a chimeric recombinant VEE/SIN alphavirus (derived from Venezuelan equine encephalitis virus [VEE] and the Sindbis virus [SIN]) encoding human immunodeficiency virus type 1 HIV-1SF162 gp140ΔV2 envelope (Env) and trimeric Env protein in MF59 adjuvant (R. Xu, I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M. Polo, S. W. Barnett, and L. Stamatatos, AIDS Res. Hum. Retroviruses 22:1022-1030, 2006). The protection did not require T-cell immune responses directed toward simian immunodeficiency virus (SIV) Gag. We extend those findings here to demonstrate antibody-mediated protection against mucosal challenge in macaques using prime-boost regimens incorporating both intramuscular and mucosal routes of delivery. The macaques in the vaccination groups were primed with VRP and then boosted with Env protein in MF59 adjuvant, or they were given VRP intramuscular immunizations alone and then challenged with SHIVSF162P4 (intrarectal challenge). The results demonstrated that these vaccines were able to effectively protect the macaques to different degrees against subsequent mucosal SHIV challenge, but most noteworthy, all macaques that received the intramuscular VRP prime plus Env protein boost were completely protected. A statistically significant association was observed between the titer of virus neutralizing and binding antibodies as well as the avidity of anti-Env antibodies measured prechallenge and protection from infection. These results highlight the merit of the alphavirus replicon vector prime plus Env protein boost vaccine approach for the induction of protective antibody responses and are of particular relevance to advancing our understanding of the potential correlates of immune protection against HIV infection at a relevant mucosal portal of entry.

Replicating Rather than Nonreplicating Adenovirus-Human Immunodeficiency Virus Recombinant Vaccines Are Better at Eliciting Potent Cellular Immunity and Priming High-Titer Antibodies

ABSTRACT A major challenge in combating the human immunodeficiency virus (HIV) epidemic is the development of vaccines capable of inducing potent, persistent cellular immunity and broadly reactive neutralizing antibody responses to HIV type 1 (HIV-1). We report here the results of a preclinical trial using the chimpanzee model to investigate a combination vaccine strategy involving sequential priming immunizations with different serotypes of adenovirus (Ad)/HIV-1MNenv/rev recombinants and boosting with an HIV envelope subunit protein, oligomeric HIVSF162 gp140ΔV2. The immunogenicities of replicating and nonreplicating Ad/HIV-1MNenv/rev recombinants were compared. Replicating Ad/HIV recombinants were better at eliciting HIV-specific cellular immune responses and better at priming humoral immunity against HIV than nonreplicating Ad-HIV recombinants carrying the same gene insert. Enhanced cellular immunity was manifested by a greater frequency of HIV envelope-specific gamma interferon-secreting peripheral blood lymphocytes and better priming of T-cell proliferative responses. Enhanced humoral immunity was seen in higher anti-envelope binding and neutralizing antibody titers and better induction of antibody-dependent cellular cytotoxicity. More animals primed with replicating Ad recombinants mounted neutralizing antibodies against heterologous R5 viruses after one or two booster immunizations with the mismatched oligomeric HIV-1SF162 gp140ΔV2 protein. These results support continued development of the replicating Ad-HIV recombinant vaccine approach and suggest that the use of replicating vectors for other vaccines may prove fruitful.

Antibody responses elicited in macaques immunized with human immunodeficiency virus type 1 (HIV-1) SF162-derived gp140 envelope immunogens : Comparison with those elicited during homologous simian/human immunodeficiency virus SHIVSF162P4 and heterologous HIV-1 infection

ABSTRACT The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIVSF162P4, and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, ΔV2gp140 (lacking the crown of the V2 loop), ΔV3gp140 (lacking the crown of the V3 loop), and ΔV2ΔV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and ΔV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and ΔV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by ΔV3gp140 or ΔV2ΔV3gp140. In contrast, the SHIVSF162P4-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.