Protection In Non-human Primates Research Articles

Protection of non-human primates against glanders with a gold nanoparticle glycoconjugate vaccine

The Gram-negative Burkholderia mallei is a zoonotic pathogen and the causative agent of glanders disease. Because the bacteria maintain the potential to be used as a biothreat agent, vaccine strategies are required for human glanders prophylaxis. A rhesus macaque (Macaca mulatta) model of pneumonic (inhalational) glanders was established and the protective properties of a nanoparticle glycoconjugate vaccine composed of Burkholderia thailandensis LPS conjugated to FliC was evaluated. An aerosol challenge dose of ∼1×104CFU B. mallei produced mortality in 50% of naïve animals (n=2/4), 2–3 days post-exposure. Although survival benefit was not observed by vaccination with a glycoconjugate glanders vaccine (p=0.42), serum LPS-specific IgG titers were significantly higher on day 80 in 3 vaccinated animals who survived compared with 3 vaccinated animals who died. Furthermore, B. mallei was isolated from multiple organs of both non-vaccinated survivors, but not from any organs of 3 vaccinated survivors at 30 days post-challenge. Taken together, this is the first time a candidate vaccine has been evaluated in a non-human primate aerosol model of glanders and represents the initial step for consideration in pre-clinical studies.

Protection of non-human primates against rabies with an adenovirus recombinant vaccine

Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials.

Genetic Variation In Vitro and In Vivo of an Attenuated Lassa Vaccine Candidate

The attenuated Lassa vaccine candidate ML29 is a laboratory-produced reassortant between Lassa and Mopeia viruses, two Old World arenaviruses that differ by 40% in nucleic acid sequence. In our previous studies, ML29 elicited sterilizing immunity against Lassa virus challenge in guinea pigs and marmosets and virus-specific cell-mediated immunity in both simian immunodeficiency virus (SIV)-infected and uninfected rhesus macaques. Here, we show that ML29 is stable after 12 passages in vitro without losing its plaque morphology or its attenuated phenotype in suckling mice. Additionally, we used deep sequencing to characterize the viral population comprising the original stock of ML29, the stock of ML29 after 12 passages in Vero cells, and the ML29 isolates obtained from vaccinated animals. Twenty-seven isolates bore approximately 77 mutations that exceeded 20% of the single-nucleotide polymorphism (SNP) changes at any single locus. Of these 77 mutations, 5 appeared to be host specific, for example, appearing in mice but not in primates. None of these mutations were reversions of ML29 to the sequences of the parental Lassa and Mopeia viruses. The host-specific mutations indicate viral adaptations to virus-host interactions, and such interactions make reasonable targets for antiviral approaches. Variants capable of chronic infection did not emerge from any of the primate infections, even in immune-deficient animals, indicating that the ML29 reassortant is reasonably stable in vivo. In conclusion, the preclinical studies of ML29 as a Lassa virus vaccine candidate have been advanced, showing high levels of protection in nonhuman primates and acceptable stability both in vitro and in vivo.

Abstract 472: Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus vaccine vectors.

Abstract Cancer vaccines continue to be of global importance. In the HIV/SIV non-human primate models replicating adenovirus has shown promise as a vaccine vector. Priming with replicating recombinant host-range mutant adenovirus (Ad5hr) followed by envelope (Env) protein boosting, elicits strong cellular, humoral and mucosal immunity, and protection in non-human primates. Based on the results using Ad5hr in the HIV/SIV non-human primate models replicating Ad4 vaccines are being developed for HIV/AIDS, and Influenza. These vectors may also be of value for cancer. A limitation of replicating Ad vectors is the transgene carrying capacity which is about 3000 base pairs (bp). Using an Ad5hr encoding full-length single-chain HIVBal gp120 linked to rhesus CD4 D1D2 domains (rhFLSC; DeVico et al., PNAS 2007) we systematically deleted the genes encoding E4-orf1 to E4-orf4. The latter virus with a 1100 bp deletion expands the total carrying capacity to ∼4000 bp. Using plaque assays we were unable to determine any differences in viral progeny production that could be attributed to the deletions. We assessed the virus-host interactions by evaluating the cell cycle profile of infected cells over time. Here too no differences could be attributed to the deletions. The Ad replication cycle is divided into early and late phases with regard to viral DNA synthesis. Western blots confirmed that the parental and deleted viruses expressed similar transgene levels in a temporally regulated manner similar to Ad late proteins. Splenocytes and sera from mice vaccinated with the parental, the E4-deleted variants, or with an Ad5hr empty vector at 0 and 4 weeks were collected at week 6 and used to evaluate whether the deletions altered transgene or vector immunogenicity. Levels of Env-specific cytokines responses by T-cells were negligible for mice immunized with the empty vector yet positive for those immunized with the parental and E4-deleted viruses. Ad responses were similar in all 6 groups. Interestingly, while the 5 immunization groups yielded similar binding antibody titers against rhFLSC and gp120 the sera showed differential binding patterns for Ad antigens. Thus while little to no host-cell or cellular immunogenicity differences distinguish the E4 deleted viruses from the parent, the differential binding patterns for Ad antigens opens the possibility that the E4 gene products may influence Ad antigen recognition. These differences may prove useful in the design of replicating Ad vaccine vectors. Citation Format: Michael A. Thomas, Rui Song, Marjorie Robert-Guroff. Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus vaccine vectors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2013-472

Immune Modulation through 4-1BB Enhances SIV Vaccine Protection in Non-Human Primates against SIVmac251 Challenge

Costimulatory molecules play a central role in the development of cellular immunity. Understanding how costimulatory pathways can be directed to positively influence the immune response may be critical for the generation of an effective HIV vaccine. Here, we evaluated the ability of intravenous administration of a blocking monoclonal antibody (mAb) directed against the negative costimulatory molecule CTLA-4, and an agonist mAb directed against the positive costimulatory molecule 4-1BB, either alone or in combination, to augment intramuscular SIV DNA immunizations. We then tested the ability these of these responses to impact a high-dose SIVmac251 challenge. Following immunization, the groups infused with the anti-4-1BB mAb exhibited enhanced IFN-γ responses compared to the DNA vaccine only group. Interestingly, although CTLA-4 blockade alone did not enhance IFN-γ responses it did increase the proliferative capacity of the CD4+ and CD8+ T cells. The combination of both mAbs enhanced the magnitude of the polyfunctional CD8+ T cell response. Following challenge, the group that received both mAbs exhibited a significant, ∼2.0 log, decrease in plasma viral load compared to the naïve group the included complete suppression of viral load in some animals. Furthermore, the use of the CTLA-4 blocking antibody resulted in significantly higher viral loads during chronic infection compared to animals that received the 4-1BB mAb, likely due to the higher CD4+ T cell proliferative responses which were driven by this adjuvant following immunization. These novel studies show that these adjuvants induce differential modulation of immune responses, which have dramatically different consequences for control of SIV replication, suggesting important implications for HIV vaccine development.

Synergistic Silencing: Combinations of Lipid-like Materials for Efficacious siRNA Delivery

Despite the promise of RNA interference (RNAi) therapeutics, progress toward the clinic has been slowed by the difficulty of delivering short interfering RNA (siRNA) into cellular targets within the body. Nearly all siRNA delivery vehicles developed to date employ a single cationic or ionizable material. In order to increase the material space available for development of siRNA delivery therapeutics, this study examined the possibility of using binary combinations of ionizable lipid-like materials to synergistically achieve gene silencing. Interestingly, it was found that ineffective single lipid-like materials could be formulated together in a single delivery vehicle to induce near-complete knockdown of firefly luciferase and factor VII in HeLa cells and in mice, respectively. Microscopy experiments suggested that synergistic action resulted when combining materials that respectively mediated cellular uptake and endosomal escape, two important steps in the delivery process. Together, the data indicate that formulating lipid-like materials in combination can significantly improve siRNA delivery outcomes while increasing the material space available for therapeutic development. It is anticipated that this binary formulation strategy could be applicable to any siRNA delivery material in any target cell population that utilizes the two-step endosomal delivery pathway.

A dendritic cell-based assay for measuring memory T cells specific to dengue envelope proteins in human peripheral blood

Dengue envelope (E) protein is a dominant immune inducer and E protein-based vaccines elicited partial to complete protection in non-human primates. To study the immunogenicity of these vaccines in humans, an enzyme linked immunospot (ELISPOT) assay for measuring interferon gamma (IFN-γ) production was developed. Cells from two subject groups, based on dengue-exposure, were selected for assay development. The unique feature of the IFN-γ ELISPOT assay is the utilization of dendritic cells pulsed with E proteins as antigen presenting cells. IFN-γ production, ranging from 53–513 spot forming units per million peripheral blood mononuclear cells (PBMCs), was observed in dengue-exposed subjects as compared to 0–45 IFN-γ spot forming units in dengue-unexposed subjects. Further, both CD4+ and CD8+ T cells, and cells bearing CD45RO memory marker, were the major sources of IFN-γ production. The assay allowed quantification of E-specific IFN-γ-secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Results suggested that the dendritic cell-based IFN-γ assay is a useful tool for assessing immunological memory for clinical research.

Progress in filovirus vaccine development: evaluating the potential for clinical use

Marburg and Ebola viruses cause severe hemorrhagic fever in humans and nonhuman primates. Currently, there are no effective treatments and no licensed vaccines; although a number of vaccine platforms have proven successful in animal models. The ideal filovirus vaccine candidate should be able to provide rapid protection following a single immunization, have the potential to work postexposure and be cross-reactive or multivalent against all Marburg virus strains and all relevant Ebola virus species and strains. Currently, there are multiple platforms that have provided prophylactic protection in nonhuman primates, including DNA, recombinant adenovirus serotype 5, recombinant human parainfluenza virus 3 and virus-like particles. In addition, a single platform, recombinant vesicular stomatitis virus, has demonstrated both prophylactic and postexposure protection in nonhuman primates. These results demonstrate that achieving a vaccine that is protective against filoviruses is possible; the challenge now is to prove its safety and efficacy in order to obtain a vaccine that is ready for human use.

Are we any closer to combating Ebola infections?

Are we any closer to combating Ebola infections?

Protection of nonhuman primates against two species of Ebola virus infection with a single complex adenovirus vector.

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.

Electroporation of Synthetic DNA Antigens Offers Protection in Nonhuman Primates Challenged with Highly Pathogenic Avian Influenza Virus

Avian influenza highlights the need for novel vaccination techniques that would allow for the rapid design and production of safe and effective vaccines. An ideal platform would be capable of inducing both protective antibodies and potent cellular immune responses. These potential advantages of DNA vaccines remain unrealized due to a lack of efficacy in large animal studies and in human trials. Questions remain regarding the potential utility of cellular immune responses against influenza virus in primates. In this study, by construct optimization and in vivo electroporation of synthetic DNA-encoded antigens, we observed the induction of cross-reactive cellular and humoral immune responses individually capable of providing protection from influenza virus infection in the rhesus macaque. These studies advance the DNA vaccine field and provide a novel, more tolerable vaccine with broad immunogenicity to avian influenza virus. This approach appears important for further investigation, including studies with humans.

Oestrogen plays a permissive role in cardioprotection

Ischaemic preconditioning (IPC) is a powerful cardioprotective intervention. It can salvage upwards of 75% of the myocardium destined to otherwise infarct. This potent cardioprotective strategy has been observed in all animal species examined to date including mammals and birds.1 We have observed IPC's protection in non-human primates (unpublished observation) and strong evidence exists of its efficacy in man.2,3 Since the initial description of IPC in 1986 by Murry et al. ,4 many investigators have toiled to uncover its mechanism in the hopes of being able to apply the strategy clinically. As a result, much is now known concerning its mechanism.5 The protection results from signal transduction pathways emanating from surface receptors. During the preconditioning ischaemia, the receptor agonists bradykinin, opioid, and adenosine are released. The first two bind to Gi-coupled receptors that trigger a complex cascade that involves nitric oxide production and opening of potassium channels in the mitochondria with subsequent redox signalling, which activates an important signalling molecule called protein kinase C (PKC). Adenosine, the third receptor agonist released by ischaemic myocardium, binds to its Gi-coupled receptor, but its signalling bypasses the above … *Corresponding author. Tel: +1 251 460 6812; fax: +1 251 460 6386. E-mail address: mcohen{at}usouthal.edu

Mucosa and vaccine-induced immune protection in nonhuman primates

The purpose of this article is to review some of the recent progress in the development of HIV vaccines that induce immune responses in mucosal tissues that may correlate with protection of the mucosal CD4 T cell compartment. Mucosal tissues are the primary sites for viral entry and the resident CD4 T cells serve as readily available target cells for active infection. Viral entry is associated with a massive destruction of these cells. The resident CD4 cells are memory T cells and hence play an important role in the generation of secondary immune responses. Protecting these CD4 T cells is critical for controlling viral infection and for preserving immune competence. Numerous mucosal vaccine regimens currently under development have been shown to induce both B and T cell responses in mucosal tissues. Though induction of neutralizing antibodies still remains an elusive goal, the demonstration that antibodies can have a protective role through alternative mechanisms offers hope that humoral responses can be harnessed to yield a protective response in mucosal tissues. The mucosal immune system is highly compartmentalized and hence immunization regimens need to target mucosal tissues to be successful in inducing protective immune responses in mucosal tissues.

Advances in the development of vaccines against Marburg and Ebola viruses.

Peter L Collins & Alexander Bukreyev† †Author for correspondence Laboratory of Infectious Disease, National Institute of Allergy & Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6505, Bethesda, MA 20892-8007, USA Tel.: +1 301 594 1854; Fax: +1 301 496 8312; ab176v@nih.gov ‘The development of vaccines against MARV and EBOV ... is a high priority because of their high virulence, ability to spread from person to person and the possibility of their use in bioterrorism.’

New HIV vaccine center has Haynes at its helm

The new Center for HIV/AIDS Vaccine Immunology (CHAVI), set up by the NIH’s National Institute of Allergy and Infectious Diseases (NIAID), is a consortium of researchers at various universities who will collaborate with the goal of creating an effective HIV vaccine that can be used worldwide. Barton Haynes, a Duke University immunologist and director of the Duke Human Vaccine Institute, is CHAVI’s new director. Haynes will work with foremost investigators Norman Letvin, Joseph Sodroski, George Shaw, and Andrew McMichael. Together they competed with and defeated 3 other potential teams to form the scientific leadership group for CHAVI, responsible for the overall work the consortium conducts. The JCI spoke to Haynes about his new position, the future of AIDS vaccines, and how this multimillion dollar endeavor might change the course of the AIDS epidemic. JCI: How was your team selected for this position? Haynes: We were selected through a peer review process of an NIH-funded grant. JCI: How will CHAVI be different from other groups that carry out AIDS vaccine research? Haynes: CHAVI will be more highly coordinated and will synergize with other groups in open and transparent ways. We will be an integral part of the overall effort of the Division of AIDS at NIAID and the Global HIV AIDS Vaccine Enterprise. JCI: What do you think are the biggest challenges that AIDS researchers face today? Haynes: Understanding correlates of protective immunity, overcoming HIV diversity, and [achieving] induction of broadly neutralizing antibodies. JCI: What do you anticipate will be your biggest challenge in this new endeavor? Haynes: The first challenge will be to establish the infrastructure for a functional, virtual organization. This will need to include a communications system of phone, video conferencing, and face-to-face meetings to develop and orchestrate functional research teams. Second, the initial focus of research for the CHAVI teams will be an extensive analysis of both the host and the virus during the very earliest stages of acute HIV infection. We will work to enroll a large cohort of acute HIV-infected patients for these studies. Third will be the analysis of those subjects repeatedly exposed to HIV who still appear to be uninfected. We will need to determine if they are truly uninfected, and if so, define the immunologic or genetic factors that contribute to protection. Next will be the challenge to define correlates of protection in nonhuman primates to a virus challenge that is relevant to transmission of HIV in human infection. And finally will be the challenge to translate our discovery efforts to the development of novel vaccine candidates for iterative testing in human trials. JCI: How do you foresee your typical day? Haynes: Lots of conference calls, visits to collaborators and advisors, lots of data analysis. Initially, we will spend time setting up administrative infrastructure, and then after the first 4-6 months, will spend more time on technology development and scientific project administration. We have already assembled a crack administrative team that knows how to run such a large and diverse organization. JCI: What are your 3 top initial goals for CHAVI? Haynes: Establishing a functional CHAVI organization infrastructure, elucidating the correlates of protective immunity in the context of acute HIV infection and primate infection models, and bringing HIV vaccine candidates to phase I clinical trials in man that, in preclinical studies, induce immune responses at systemic and mucosal sites, and that we believe will be predictive of clinically relevant protective anti-HIV immune responses in man. JCI: What in your life are you most proud of? Haynes: My family. And coaching my children’s sports teams.

Efficacy of AIDS vaccine strategies in nonhuman primates

Since only a limited number of vaccines can be tested for efficacy in phase 3 studies in humans, a filter is needed allowing selection of the most promising ones. Although differences between HIV infection in humans and simian immunodeficiency virus infection in nonhuman primates (NHP) might limit the predictive value of these models, comparative efficacy studies in NHPs could facilitate ranking of vaccine candidates. While various forms of protein vaccines failed to induce consistent protection, live-attenuated vaccines, DNA vaccines and viral vector vaccines provided various levels of protection in NHPs. However, variability in the experimental models limits the conclusions that can be drawn with respect to the relative efficacy of vaccines not tested in the same experiment. Therefore, better standardization is an urgent necessity in order to exploit the full potential of nonhuman primate models in AIDS vaccine development.

Mucosal delivery of the human immunodeficiency virus-1 Tat protein in mice elicits systemic neutralizing antibodies, cytotoxic T lymphocytes and mucosal IgA

Human immunodeficiency virus (HIV)-1 Tat protein induces protection in non-human primates upon systemic vaccination. In view of the design of mucosal vaccines against HIV-1 we studied the immune response to native Tat (aa 1–86) in mice following intranasal delivery of the protein with two mucosal adjuvants, Escherichia coli heat-labile enterotoxin (LT) and LT-R72, a non-toxic mutant of LT. Immunization with Tat and the two adjuvants induced in BALB/c but not in C57BL/6 mice high and persistent levels of serum IgG and secretory IgA in vaginal and intestinal fluids. Mice sera neutralized Tat and recognized two epitopes mapping in the regions 1–20 and 46–60. Furthermore, their splenocytes proliferated and secreted IFN-γ and IL-6 in response to Tat. Finally, CTLs were also elicited and they recognized an epitope localized within aa 11–40 of Tat.

Antimicrobial strategies in the prevention of dental caries.

Antimicrobial agents, applied either professionally or delivered from dentifrices or mouthwashes, could reduce caries by controlling plaque formation, suppressing cariogenic species, or by inhibiting bacterial metabolism. Chlorhexidine has proven anticaries activity; other agents might also prevent caries by means of their antiplaque properties. Sugar substitutes stabilize the microflora by reducing the number of acid challenges to plaque and stimulating saliva flow; some also have antimicrobial properties, especially against mutans streptococci. Vaccines have been prepared from purified antigens of mutans streptococci. These vaccines confer protection in non-human primates, but have yet to be tested in a human clinical trial.