Live attenuated HIV vaccines: Predicting the tradeoff between efficacy and safety

The utility of live attenuated vaccines for controlling HIV epidemics is being debated. Live attenuated HIV vaccines (LAHVs) could be extremely effective in protecting against infection with wild-type strains, but may not be completely safe as the attenuated strain could cause AIDS in some vaccinated individuals. We present a theoretical framework for evaluating the consequences of the tradeoff between vaccine efficacy (in terms of preventing new infections with wild-type strains) and safety (in terms of vaccine-induced AIDS deaths). We use our framework to predict, for Zimbabwe and Thailand, the epidemiological impact of 1,000 different (specified by efficacy and safety characteristics) LAHVs. We predict that paradoxically: (i) in Zimbabwe (where transmission is high) LAHVs would significantly decrease the AIDS death rate, but (ii) in Thailand (where transmission is low) exactly the same vaccines (in terms of efficacy and safety characteristics) would increase the AIDS death rate. Our results imply that a threshold transmission rate exists that determines whether any given LAHV has a beneficial or a detrimental impact. We also determine the vaccine perversity point, which is defined in terms of the fraction of vaccinated individuals who progress to AIDS as a result of the vaccine strain. Vaccination with any LAHV that causes more than 5% of vaccinated individuals to progress to AIDS in 25 years would, even 50 years later, lead to perversity (i.e., increase the annual AIDS death rate) in Thailand; these same vaccines would lead to decreases in the annual AIDS death rate in Zimbabwe.

A candidate live inactivatable attenuated vaccine for AIDS.

The recent discovery of long term AIDS nonprogressors who harbor nef-attenuated HIV suggests that a naturally occurring live vaccine for AIDS may already exist. Animal models have shown that a live vaccine for AIDS, attenuated in nef, is the best candidate vaccine. There are considerable risks, real and perceived, with the use of live HIV vaccines. We have introduced a conditional lethal genetic element into HIV-1 and simian immunodeficiency virus (SIV) molecular clones deleted in nef. The antiviral strategy we employed targets both virus replication and the survival of the infected cell. The suicide gene, herpes simplex virus thymidine kinase (tk), was expressed and maintained in HIV over long periods of time. Herpes simplex virus tk confers sensitivity to the antiviral activity of acyclic nucleosides such as ganciclovir (GCV). HIV-tk and SIV-tk replication were sensitive to GCV at subtoxic concentrations, and virus-infected cells were eliminated from tumor cell lines as well as primary cell cultures. We found the HIV-tk virus to be remarkably stable even after being cultured in media containing a low concentration of GCV and then challenged with the higher dose and that while GCV resistant escape mutants did arise, a significant fraction of the virus remained sensitive to GCV.

Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.

CTXφ immunity: Application in the development of cholera vaccines

CTXφ is a filamentous bacteriophage that encodes cholera toxin, the principal virulence factor of Vibrio cholerae. CTXφ is unusual among filamentous phages because it encodes a repressor and forms lysogens. CTXφ can infect the existing live-attenuated V. cholerae vaccine strains derived from either the El Tor or classical V. cholerae biotypes and result in vaccine reversion to toxinogenicity. Intraintestinal CTXφ transduction assays were used to demonstrate that El Tor biotype strains of V. cholerae are immune to infection with the El Tor-derived CTXφ, whereas classical strains are not. The El Tor CTXφ repressor, RstR, was sufficient to render classical strains immune to infection with the El Tor CTXφ. The DNA sequences of the classical and El Tor CTXφ repressors and their presumed cognate operators are highly diverged, whereas the sequences that surround this “immunity” region are nearly identical. Transcriptional fusion studies revealed that the El Tor RstR mediated repression of an El Tor rstA-lacZ fusion but did not repress a classical rstA-lacZ fusion. Likewise, the classical RstR only repressed a classical rstA-lacZ fusion. Thus, similar to the mechanistic basis for heteroimmunity among lambdoid phages, the specificity of CTXφ immunity is based on the divergence of the sequences of repressors and their operators. Expression of the El Tor rstR in either El Tor or classical live-attenuated V. cholerae vaccine strains effectively protected these vaccines from CTXφ infection. Introduction of rstR into V. cholerae vaccine strains should enhance their biosafety.

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVΔpac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVΔpac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVΔpac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVΔpac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVΔpac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD50. Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Endocrine modulation of the neurotoxicity of gp120: Implications for AIDS-related dementia complex

HIV infection often involves the development of AIDS-related dementia complex, a variety of neurologic, neuropsychologic, and neuropathologic impairments. A possible contributor to AIDS-related dementia complex is the HIV envelope glycoprotein gp120, which damages neurons via a complex glutamate receptor- and calcium-dependent cascade. We demonstrate an endocrine modulation of the deleterious effects of gp120 in primary hippocampal and cortical cultures. Specifically, we observe that gp120-induced calcium mobilization and neurotoxicity are exacerbated by glucocorticoids, the adrenal steroids secreted during stress. Importantly, this deleterious synergy can occur between gp120 and synthetic glucocorticoids (such as prednisone or dexamethasone) that are used clinically in high concentrations to treat severe cases of the Pneumocystis carinii pneumonia typical of HIV infection. Conversely, we also observe that estradiol protects neurons from the deleterious actions of gp120, reducing toxicity and calcium mobilization.

CD68+ cells of monocyte/macrophage lineage in the environment of AIDS-associated and classic-sporadic Kaposi sarcoma are singly or doubly infected with human herpesviruses 7 and 6B

Earlier studies have shown that Kaposi sarcomas contain cells infected with human herpesvirus (HHV) 6B, and in current studies we report that both AIDS-associated and classic-sporadic Kaposi sarcoma contain HHV-7 genome sequences detectable by PCR. To determine the distribution of HHV-7-infected cells relative to those infected with HHV-6, sections from paraffin-embedded tissues were allowed to react with antibodies to HHV-7 virion tegument phosphoprotein pp85 and to HHV-6B protein p101. The antibodies are specific for HHV-7 and HHV-6B, respectively, and they retained reactivity for antigens contained in formalin-fixed, paraffin-embedded tissue samples. We report that (i) HHV-7 pp85 was present in 9 of 32 AIDS-associated Kaposi sarcomas, and in 1 of 7 classical-sporadic HIV-negative Kaposi sarcomas; (ii) HHV-7 pp85 was detected primarily in cells bearing the CD68 marker characteristic of the monocyte/macrophage lineage present in or surrounding the Kaposi sarcoma lesions; and (iii) in a number of Kaposi sarcoma specimens, tumor-associated CD68+ monocytes/macrophages expressed simultaneously antigens from both HHV-7 and HHV-6B, and therefore appeared to be doubly infected with the two viruses. CD68+ monocytes/macrophages infected with HHV-7 were readily detectable in Kaposi sarcoma, but virtually absent from other normal or pathological tissues that harbor macrophages. Because all of the available data indicate that HHV-7 infects CD4+ T lymphocytes, these results suggest that the environment of the Kaposi sarcoma (i) attracts circulating peripheral lymphocytes and monocytes, triggers the replication of latent viruses, and thereby increases the local concentration of viruses, (ii) renders CD68+ monocytes/macrophages susceptible to infection with HHV-7, and (iii) the combination of both events enables double infections of cells with both HHV-6B and HHV-7.

Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines

We have reported previously that murine bone marrow-derived dendritic cells (DC) pulsed with whole tumor lysates can mediate potent antitumor immune responses both in vitro and in vivo. Because successful therapy was dependent on host immune T cells, we have now evaluated whether the systemic administration of the T cell stimulatory/growth promoting cytokine interleukin-2 (IL-2) could enhance tumor lysate-pulsed DC-based immunizations to further promote protective immunity toward, and therapeutic rejection of, syngeneic murine tumors. In three separate approaches using a weakly immunogenic sarcoma (MCA-207), the systemic administration of nontoxic doses of recombinant IL-2 (20,000 and 40,000 IU/dose) was capable of mediating significant increases in the potency of DC-based immunizations. IL-2 could augment the efficacy of tumor lysate-pulsed DC to induce protective immunity to lethal tumor challenge as well as enhance splenic cytotoxic T lymphocyte activity and interferon-γ production in these treated mice. Moreover, treatment with the combination of tumor lysate-pulsed DC and IL-2 could also mediate regressions of established pulmonary 3-day micrometastases and 7-day macrometastases as well as established 14- and 28-day s.c. tumors, leading to either significant cure rates or prolongation in overall survival. Collectively, these findings show that nontoxic doses of recombinant IL-2 can potentiate the antitumor effects of tumor lysate-pulsed DC in vivo and provide preclinical rationale for the use of IL-2 in DC-based vaccine strategies in patients with advanced cancer.

β-Chemokines and neutralizing antibody titers correlate with sterilizing immunity generated in HIV-1 vaccinated macaques

One of the obstacles to AIDS vaccine development is the variability of HIV-1 within individuals and within infected populations, enabling viral escape from highly specific vaccine induced immune responses. An understanding of the different immune mechanisms capable of inhibiting HIV infection may be of benefit in the eventual design of vaccines effective against HIV-1 variants. To study this we first compared the immune responses induced in Rhesus monkeys by using two different immunization strategies based on the same vaccine strain of HIV-1. We then utilized a chimeric simian/HIV that expressed the envelope of a dual tropic HIV-1 escape variant isolated from a later time point from the same patient from which the vaccine strain was isolated. Upon challenge, one vaccine group was completely protected from infection, whereas all of the other vaccinees and controls became infected. Protected macaques developed highest titers of heterologous neutralizing antibodies, and consistently elevated HIV-1-specific T helper responses. Furthermore, only protected animals had markedly increased concentrations of RANTES, macrophage inflammatory proteins 1α and 1β produced by circulating CD8+ T cells. These results suggest that vaccine strategies that induce multiple effector mechanisms in concert with β-chemokines may be desired in the generation of protective immune responses by HIV-1 vaccines.

Identification of inflections in T-cell counts among HIV-1-infected individuals and relationship with progression to clinical AIDS

Studies of circulating T (CD3+) lymphocytes have shown that on a population basis T-cell numbers remain stable for many years after HIV-1 infection (blind T-cell homeostasis), but decline rapidly beginning approximately 1.5–2.5 years before the onset of clinical AIDS. We derived a general method for defining the loss of homeostasis on the individual level and for determining the prevalence of homeostasis loss according to HIV status and the occurrence of AIDS in more than 5,000 men enrolled in the Multicenter AIDS Cohort Study. We used a segmented regression model for log10 CD3+ cell counts that included separate T-cell trajectories before and after a time (the T-cell inflection point) where the loss of T-cell homeostasis was most likely to have occurred. The average slope of CD3+ lymphocyte counts before the inflection point was close to zero for HIV− and HIV+ men, consistent with blind T-cell homeostasis. After the inflection point, the HIV+ individuals who developed AIDS generally showed a dramatic decline in CD3+ cell counts relative to HIV− men and HIV+ men not developing AIDS. A CD3+ cell decline of greater than 10 percent per year was present in 77% of HIV+ men developing AIDS but in only 23% of HIV+ men with no onset of AIDS. Our findings at the individual level support the blind T-cell homeostasis hypothesis and provide strong evidence that the loss of homeostasis is an important mechanism in the pathogenesis of the severe immunodeficiency that characterizes the late stages of HIV infection.

Are the pneumococcal polysaccharide vaccines effective? Meta-analysis of the prospective trials

The objective was to review the evidence of effectiveness of the polyvalent polysaccharide pneumococcal vaccine from prospective properly randomised controlled trials comparing pneumococcal vaccines with placebo in subjects who are immunocompetent and those likely to have an impaired immune system.

Chronic elevation of plasma thioredoxin: Inhibition of chemotaxis and curtailment of life expectancy in AIDS

Thioredoxin (Trx) is an intracellular redox protein with extracellular cytokine-like and chemokine-like activities. We show here that, although plasma Trx levels are unrelated to survival of HIV-infected individuals with CD4 cell counts above 200/μl blood, survival is significantly impaired (P = 0.003) when plasma Trx is chronically elevated in HIV-infected subjects with CD4 T cell counts below this level (i.e., with Centers for Disease Control (CDC)-defined AIDS). Relevant to the mechanism potentially underlying this finding, we also present data from experimental studies in mice showing that elevated plasma Trx efficiently blocks lipopolysaccharide (LPS)-induced chemotaxis, an innate immune mechanism that is particularly crucial when adaptive immunity is compromised. Thus, we propose that elevated plasma Trx in HIV-infected individuals with low CD4 T cell counts directly impairs survival by blocking pathogen-induced chemotaxis, effectively eliminating the last (innate) barrier against establishment of opportunistic and other infections in these immunodeficient individuals.

Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, carrier structure, and adjuvant all influence antibody responses to Lewisy conjugates in mice

The feasibility of using carbohydrate-based vaccines for the immunotherapy of cancer is being actively explored at the present time. Although a number of clinical trials have already been conducted with glycoconjugate vaccines, the optimal design and composition of the vaccines has yet to be determined. Among the candidate antigens being examined is Lewisy (Ley), a blood group-related antigen that is overexpressed on the majority of human carcinomas. Using Ley as a model for specificity, we have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Ley conjugates in mice. A glycolipopeptide containing a cluster of three contiguous Ley-serine epitopes and the Pam3Cys immunostimulating moiety was found to be superior to a similar construct containing only one Ley-serine epitope in eliciting antitumor cell antibodies. Because only IgM antibodies were produced by this vaccine, the effect on immunogenicity of coupling the glycopeptide to keyhole limpet hemocyanin was examined; although both IgM and IgG antibodies were formed, the antibodies reacted only with the immunizing structure. Reexamination of the clustered Ley-serine Pam3Cys conjugate with the adjuvant QS-21 resulted in the identification of both IgG and IgM antibodies reacting with tumor cells, thus demonstrating the feasibility of an entirely synthetic carbohydrate-based anticancer vaccine in an animal model.