Comparative studies of Avipox-GM-CSF versus recombinant GM-CSF protein as immune adjuvants with different vaccine platforms

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immune stimulant when administered with different vaccines. Optimal use of GM-CSF resides in its ability to act locally to stimulate the proliferation and maturation of professional antigen-presenting cells (APCs) (i.e., Langerhans' cells) at the injection site. GM-CSF was engineered into a replication-incompetent recombinant avian (fowlpox) virus (rF-GM-CSF) and a single subcutaneous injection resulted in a sustained enrichment of activated dendritic cells within the regional draining lymph nodes. Those changes were attributed to local GM-CSF production at the injection site by rF-GM-CSF-infected cells. Studies were carried out in which mice were administered different types of beta-galactosidase (beta-gal)-based vaccines--whole protein, peptide, recombinant poxviruses--and GM-CSF was administered either as a single injection of rF-GM-CSF or four daily bolus injections of the recombinant protein. The use of rF-GM-CSF either improved the immune adjuvant effect, as observed for poxvirus-based vaccines, or was equivalent to rGM-CSF, as observed with the beta-gal protein vaccine. It is important to note that with either the replication-competent (vaccinia) or replication-incompetent (fowlpox) vaccines expressing LacZ, strong CTL responses directed against beta-gal were induced only when rF-GM-CSF was used as the immune adjuvant. Engineering GM-CSF into a recombinant fowlpox virus offers an excellent vehicle for the delivery of this cytokine as an immune adjuvant with specific vaccine platforms. In particular, delivery of GM-CSF via the rF-GM-CSF construct would be preferred over bolus injections of rGM-CSF when used as an immune adjuvant with whole protein or recombinant poxvirus-based vaccines. The study underscores the importance of defining the appropriate delivery form of an immune adjuvant, such as GM-CSF, relative to the immunization strategy to maximize the host immune responses against a specific antigen.     

The effectiveness in rhesus monkeys of an antifertility vaccine based on neutralization of chorionic gonadotropin

A model system, employing non-human primates, is needed to test the postulate that active immunization to achieve neutralization of the biological activity of chorionic gonadotropin could be used as a basis for fertility regulation in women. Adult female rhesus monkeys were actively immunized with the β-subunit of ovine LH (oLHβ) mixed with Freund's complete adjuvant. All monkeys developed circulating antibodies which bound iodinated oLH and human chorionic gonadotropin (hCG). The bound hCG was displaced by rhesus chorionic gonadotropin (rhCG). Antisera were also able to neutralize the biological activity of rhCG in the immature rat uterine weight assay. Serum progesterone levels and menstrual bleeding patterns indicated that normal ovulatory cycles continued to occur in most cases after immunization. Results of mating the animals reveal that the fertility of the immunized animals was significantly reduced.     

A computational resource for the prediction of peptide binding to Indian rhesus macaque MHC class I molecules

Non-human primates, in general, and Indian rhesus macaques, specifically, play an important role in the development and testing of vaccines and diagnostics destined for human use. To date, several frequently expressed macaque MHC molecules have been identified and their binding specificities characterized in detail. Here, we report the development of computational algorithms to predict peptide binding and potential T cell epitopes for the common MHC class I alleles Mamu-A*01, -A*02, -A*11, -B*01 and -B*17, which cover approximately two thirds of the captive Indian rhesus macaque populations. We validated this method utilizing an SIV derived data set encompassing 59 antigenic peptides. Of all peptides contained in the SIV proteome, the 2.4% scoring highest in the prediction contained 80% of the antigenic peptides. The method was implemented in a freely accessible and user friendly website at . Thus, we anticipate that our approach can be utilized to rapidly and efficiently identify CD8+ T cell epitopes recognized by rhesus macaques and derived from any pathogen of interest.     

Modulation of antigen-specific cellular immune responses to DNA vaccination in rhesus macaques through the use of IL-2, IFN-gamma, or IL-4 gene adjuvants

Extensive experiments have shown DNA vaccines' ability to elicit immune responses in vivo in a safe and well-tolerated manner in several model systems, including rodents and non-human primates. As the DNA-based vaccine and immunotherapy approaches are being explored in humans, significant efforts have also been focused on further improving the immune potency of this technology. One strategy to enhance immune responses for DNA vaccines is the use of molecular or genetic adjuvants. These molecular adjuvant constructs (which encodes for immunologically important molecules such as cytokines) can be co-administered along with DNA vaccine constructs. Once delivered, these adjuvants have shown to modulate the magnitude and direction (humoral or cellular) of the vaccine-induced immune responses in rodent models. To date, however, there has been very little data reported from studies in primates. In this study, we examined the effects of cytokine gene adjuvants to enhance the level of cell-mediated immune responses in rhesus macaques. We co-immunized rhesus macaques with expression plasmids encoding for IL-2, IFN-gamma or IL-4 cytokines along with the DNA vaccine constructs encoding for HIV env/rev (pCEnv) and SIV gag/pol (pCSGag/pol) proteins. We observed that coadministration of IL-2 and IFN-gamma cDNA resulted in enhancement of antigen-specific T cell-mediated immune responses.     

Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques

DNA vaccines are a promising technology. Historically, however, the ability of DNA vaccines to induce high response rates and strong immune responses, especially antibody responses, in non-human primates and human clinical trials has proven suboptimal. Here, we performed a pilot study in rhesus macaques to evaluate whether we could improve the immunogenicity of DNA vaccines through the use of adjuvant technology and improved delivery systems. The study consisted of four groups of animals that received: DNA by intramuscular (IM) injection, DNA with plasmid-encoded IL-12 by IM injection, DNA by IM injection with in vivo electroporation (EP), and DNA with IL-12 by IM EP. Each group was immunized three times with optimized HIV gag and env constructs. Vaccine immunogenicity was assessed by IFNgamma ELISpot, CFSE proliferation, polyfunctional flow cytometry, and antibody ELISA. Similar to previous studies, use of IL-12 as an adjuvant increased the gag and env-specific cellular responses. The use of EP to enhance plasmid delivery resulted in dramatically higher cellular as well as humoral responses. Interestingly, the use of EP to administer the DNA and IL-12 adjuvant combination resulted in the induction of higher, more efficient responses such that a 10-fold increase in antigen-specific IFNgamma(+) cells compared to IM DNA immunization was observed after a single immunization. In addition to increases in the magnitude of IFNgamma production in the initial and memory responses, the combined approach resulted in enhancements in the proliferative capacity of antigen-specific CD8(+) T cells and the amount of polyfunctional cells capable of producing IL-2 and TNFalpha in addition to IFNgamma. These data suggest that adjuvant and improved delivery methods may be able to overcome previous immunogenicity limitations in DNA vaccine technology.     

Immunogenicity and performance of an enterovirus 71 virus-like-particle vaccine in nonhuman primates

A vaccine against human enterovirus 71 (EV-A71) is urgently needed to combat outbreaks of EV-A71 and in particular, the serious neurological complications that manifest during these outbreaks. In this study, an EV-A71 virus-like-particle (VLP) based on a B5 subgenogroup (EV-A71-B5 VLP) was generated using an insect cell/baculovirus platform. Biochemical analysis demonstrated that the purified VLP had a highly native procapsid structure and initial studies in vivo demonstrated that the VLPs were immunogenic in mice. The impact of VLP immunization on infection was examined in non-human primates using a VLP prime-boost strategy prior to EV-A71 challenge. Rhesus macaques were immunized on day 0 and day 21 with VLPs (100 μg/dose) containing adjuvant or with adjuvant alone (controls), and were challenged with EV-A71 on day 42. Complete blood counts, serum chemistry, magnetic resonance imaging (MRI) scans, and histopathology results were mostly normal in vaccinated and control animals after virus challenge demonstrating that the fatal EV-A71-B3 clinical isolate used in this study was not highly virulent in rhesus macaques. Viral genome and/or infectious virus were detected in blood, spleen or brain of two of three control animals, but not in any specimens from the vaccinated animals, indicating that VLP immunization prevented systemic spread of EV-A71 in rhesus macaques. High levels of IgM and IgG were detected in VLP-vaccinated animals and these responses were highly specific for EV-A71 particles and capsid proteins. Serum from vaccinated animals also exhibited similar neutralizing activity against different subgenogroups of EV-A71 demonstrating that the VLPs induced cross-neutralizing antibodies. In conclusion, our EV-A71-B5 VLP is safe, highly immunogenic, and prevents systemic EV-A71-B3 infection in nonhuman primates making it a viable attractive vaccine candidate for EV-A71.     

Comparison of T cell immune responses induced by vectored HIV vaccines in non-human primates and humans

Following the disappointing outcome of the phase IIb test-of-concept step study in which Merck's adenovirus type 5 (Ad5) HIV-1 clade B gag/pol/nef vaccine failed to demonstrate efficacy in HIV high-risk individuals, an extensive review of the trial and preclinical studies which supported the trial is ongoing. One point of interest is how well preclinical nonhuman primate immunogenicity studies predicted what was observed in humans. Here we compare the HIV-1-specific cellular immune responses elicited in nonhuman primates and human clinical trial subjects to several HIV-1 vaccine candidates. We find that although rhesus macaques are immunologically more responsive to vaccination than humans, the hierarchy in potency of single-modality prime-boost regimens using several vector approaches (adenovirus, DNA, and pox vectors) was well predicted. Vaccine approaches using complex formulations such as novel adjuvants (DNA+CRL1005) or mixed-modality prime-boost (DNA/Ad5; Ad5/ALVAC) did not correlate as well between rhesus macaques and humans. Although the immunogenicity of the vaccines and vaccine regimens evaluated were not all accurately predicted, testing in rhesus macaques generally offers an indispensable tool for ranking the immunological potential of HIV-1 vaccine candidates.     

Efficacy of granulocyte-macrophage colony-stimulating factor (GM-CSF) as a vaccine adjuvant for hepatitis B virus in patients with HIV infection

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a cytokine with a potential vaccine adjuvant activity. It is also known that human immunodeficiency virus (HIV) infected patients often show poor immunologic responses to immunization. We examined whether the use of GM-CSF could augment the immunologic response to recombinant vaccine against the hepatitis B virus (HBV) in 80 HIV infected patients (18-35 years old). They received a double dose (40 microg) of recombinant HBV vaccine IM at 0, 1 and 6 months and were randomized to receive either concurrent 20 microg of GM-CSF (n=40) or placebo IM (n=40) with the first vaccine dose. A significant increase in the seroconversion rate was observed after the second vaccine dose in the GM-CSF group (62% GM-CSF versus 30% control group P<0.0074). The average anti-HBs titers measured on days 28, 60 and 210 were 40.3; 366.5 and 644.8 milli-international units per milliliter (mIU/ml), respectively, in the GM-CSF group, and 62.4; 166.4 and 375.0 mIU/ml, respectively, in the control group, with significant differences at 60 and 210 days (P<0.01). There were no significant differences between CD4/CD8 cells, viral load, risk factors, age, sex and the serological responses to the HBV vaccine. This study suggests that GM-CSF increases the immunogenicity of recombinant HBV vaccine in HIV infected individuals.     

Viral vectored granulocyte-macrophage colony stimulating factor inhibits vaccine protection in an SIV challenge model: protection correlates with neutralizing antibody

In a previous vaccine study, we reported significant and apparently sterilizing immunity to high-dose, mucosal, simian immunodeficiency virus (SIV) quasi-species challenge. The vaccine consisted of vectors based on vesicular stomatitis virus (VSV) expressing simian immunodeficiency virus (SIV) gag and env genes, a boost with propagating replicon particles expressing the same SIV genes, and a second boost with VSV-based vectors. Concurrent with that published study we had a parallel group of macaques given the same doses of vaccine vectors, but in addition, we included a third VSV vector expressing rhesus macaque GM-CSF in the priming immunization only. We report here that addition of the vector expressing GM-CSF did not enhance CD8 T cell or antibody responses to SIV antigens, and almost completely abolished the vaccine protection against high-dose mucosal challenge with SIV. Expression of GM-CSF may have limited vector replication excessively in the macaque model. Our results suggest caution in the use of GM-CSF as a vaccine adjuvant, especially when expressed by a viral vector. Combining vaccine group animals from this study and the previous study we found that there was a marginal but significant positive correlation between the neutralizing antibody to a neutralization resistant SIV Env and protection from infection.     

Needle-free delivery of DNA: Targeting of hemagglutinin to MHC class II molecules protects rhesus macaques against H1N1 influenza

Conventional influenza vaccines are hampered by slow and limited production capabilities, whereas DNA vaccines can be rapidly produced for global coverage in the event of an emerging pandemic. However, a drawback of DNA vaccines is their generally low immunogenicity in non-human primates and humans. We have previously demonstrated that targeting of influenza hemagglutinin to human HLA class II molecules can increase antibody responses in larger animals such as ferrets and pigs. Here, we extend these observations by immunizing non-human primates (rhesus macaques) with a DNA vaccine encoding a bivalent fusion protein that targets influenza virus hemagglutinin (HA) to Mamu class II molecules. Such immunization induced neutralizing antibodies and antigen-specific T cells. The DNA was delivered by pain- and needle-free jet injections intradermally. No adverse effects were observed. Most importantly, the immunized rhesus macaques were protected against a challenge with influenza virus.     

Topical delivery of imiquimod to a mouse model as a novel adjuvant for human immunodeficiency virus (HIV) DNA

We evaluated the compound imiquimod as a possible adjuvant for DNA immunization against human immunodeficiency virus (HIV). We found that gene-gun epidermal delivery of the DNA in combination with imiquimod resulted in the strongest HIV specific immune responses. The effect of imiquimod was further compared to that of recombinant granulocyte macrophage-colony stimulating factor (GM-CSF), a known DNA vaccine adjuvant. Both adjuvants were able to enhance the immune responses induced by the HIV-1 genes alone. The delivery of an adjuvant as a topical cream rather than through injections has a clear clinical benefit. We show for the first time that imiquimod can act as an adjuvant for DNA vaccination.     

Intramuscular immunization with a monogenic plasmid DNA tuberculosis vaccine: Enhanced immunogenicity by electroporation and co-expression of GM-CSF transgene

Plasmid DNA vaccine has been widely explored for tuberculosis immunization but there is a need to develop the ways to improve its immunogenicity. In this study, we have constructed a plasmid DNA vaccine coding for Ag85A alone or for both Ag85A and GM-CSF and investigated the immune adjuvant effects of electroporation and GM-CSF co-expression, alone or in combination, on CD4 and CD8 T cell IFN-gamma responses, CTL activities and immune protection from pulmonary Mycobacterium tuberculosis challenge in a Balb/c mouse model. We have found that use of electroporation allows a single intramuscular (i.m.) DNA injection to be as effective as repeated i.m. DNA injections in activation of both Ag85A-specific CD4 and CD8 T cells. Co-expression of immune-enhancing cytokine GM-CSF by the same plasmid DNA TB vaccine could further enhance T cell activation including CTL activities on top of electroporation. With regard to immune protection from pulmonary M. tb challenge, use of electroporation also allows a single i.m. DNA injection to be as effective as repeated i.m. DNA injections. Co-expression of GM-CSF transgene also moderately enhances immune protection and such effect is more evident for systemic protection. However, GM-CSF expression has little added effect on immune protection by electroporation-aided immunization protocols. Our findings thus will help with the development of future DNA TB immunization strategies.     

Hormones and Sexual Behavior in the Female

Studies in rhesus monkeys suggest that female sexual receptivity depends on hormonal feedback effects on brain mechanisms, the result of which is enhanced invitational behavior. Production of pheromones by bacteria present in vaginal secretions is also hormone-dependent. This symbiosis between a mammalian and a bacterial system occurs in other subhuman primates and possibly in the human female as well.     

The reproductive endocrine system of nonhuman primates—A model for prediction of toxicity

The following statements summarize our present judgements of the areas of reproductive endocrinology in which rhesus monkeys and chimpanzees can appropriately serve as surrogates for man. In many cases these conclusions are based on preliminary data and may be altered by subsequent experiments. Because of different patterns of steroid synthesis and metabolism, the rhesus monkey is not a good model for human pregnancy. Endocrine relationships in the three species are similar during the neonatal period, but due to the lack of a detectable adrenal maturation process the rhesus monkey may not be an appropriate model for studies during puberty. Control of the menstrual cycle appears to be similar in each of the three species. While post-menopausal rhesus monkeys have been found and their hormone patterns are similar to those of postmenopausal humans, menopause has not yet been documented in chimpanzees. Studies of the relationship of the hypothalamus to the pituitary in rhesus monkeys would be difficult since they do not uniformly respond to GnRH. Preliminary results of our initial attempts to utilize the reproductive endocrine system of primates as a means of predicting toxicity have been presented. While sufficient information is not yet available to enable a definitive outline of all the facets of the primate endocrine system which are liable to specific toxic intervention, we hope this presentation will elicit the interest of both toxicologists and endocrinologists in a fruitful marriage of the two disciplines.     

Induction of a protective immunity against Schistosoma mansoni with ovalbumin-coupled Sm37-5 coadsorbed with granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-12 on alum

A previous study has shown that Sm37-5 is a major B cell epitope of Sm37-GAPDH. This epitope is highly antigenic in human infections and IgG antibody reactivity toward this determinant is associated with adolescent resistance to reinfection. This led us to test a synthetic peptide corresponding to Sm37-5, coupled to ovalbumin, as an anti-schistosome vaccine. Although mice injected with Sm37-5-OVA in Freund's adjuvant showed significant protection, immunization in aluminium hydroxide failed to induce protection. The adjuvant effect of cytokines (GM-CSF or IL-12) associated with the antigen on alum was investigated. With each of these two cytokines, significant reductions in the worm burden were obtained (32-38% with GM-CSF and 27% with IL-12, respectively). In addition, a reduction of the egg number trapped in the liver of immunized mice was also observed. Thus, protections were obtained with formulations that could potentially be used in humans.     

Novel codon-optimized GM-CSF gene as an adjuvant to enhance the immunity of a DNA vaccine against HIV-1 Gag

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine. Here we generated a novel codon-optimized murine GM-CSF gene as an adjuvant. The codon-optimized GM-CSF gene significantly increased protein expression levels in all cells tested. Although injection of the wild-type GM-CSF plasmids adjuvanted HIV-1 Gag DNA vaccine induced detectable immune responses, co-administration of plasmids encoding the codon-optimized GM-CSF sequence with the DNA vaccine resulted in a strong antibody and CTL responses and a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag. This novel codon-optimized GM-CSF gene offers a practical molecular strategy for potentiating immune responses to vaccines as well as other immunotherapeutic strategies.     

Delivery of the HIV-1 Tat protein to dendritic cells by the CyaA vector induces specific Th1 responses and high affinity neutralizing antibodies in non human primates

The human immunodeficiency virus type 1 (HIV-1) Tat is a key protein playing a major role in the infectivity of the virus. Thus, HIV-Tat based vaccines have been proposed as an attractive option to treat AIDS. Recently, we have shown that the recombinant detoxified adenylate cyclase (CyaA) from Bordetella pertussis carrying HIV-Tat (CyaA-E5-Tat), targets dendritic cells (DCs) and induces specific Th1 polarized and neutralizing antibody responses in mice. To further explore the potentialities of this prototype vaccine for human use, we analyzed the CyaA-E5-Tat induced antibody responses in non-human primates and established the biological characteristics of these antibodies. African Green Monkeys (AGM) were immunized with CyaA-E5-Tat in the presence or in the absence of alum adjuvant. First, we showed that the anti-CyaA antibodies induced by such immunization does not interfere with the binding of CyaA-E5-Tat to its receptor at the DC surface, the alphaMbeta2 integrin. Monkeys immunized with CyaA-E5-Tat, with or without alum, produced anti-Tat antibodies that mainly recognized the N-terminal domain of the Tat protein. Importantly, all sera obtained after three immunizations displayed the capacity to bind to Tat and neutralize its transactivating function in vitro. Finally, in the absence of alum, CyaA-E5-Tat, induced Th1 Tat specific T cell responses. These findings reveal that CyaA-E5-Tat is efficiently delivered in non-human primates and had a significant impact on the generation of neutralizing anti-Tat antibodies. These observations are, thus, encouraging for the use of the CyaA vector in human and also suggest that CyaA-E5-Tat might be a useful tool to decipher the biological characteristic of such antibodies.     

Intradermally administered TLR4 agonist GLA-SE enhances the capacity of human skin DCs to activate T cells and promotes emigration of Langerhans cells

The natural TLR4 agonist lipopolysaccharide (LPS) has notable adjuvant activity. However, it is not useful as a vaccine adjuvant due to its toxicity. Glucopyranosyl lipid A (GLA) is a synthetic derivative of the lipid A tail of LPS with limited cytotoxicity, but strong potential to induce immune responses in mice, guinea pigs, non-human primates, and humans. In this study we determined how this synthetic TLR4 agonist affects the function of different subsets of human skin dendritic cells (DCs). The effect of GLA in an aqueous formulation (GLA-AF) or in an oil-in-water emulsion (GLA-SE) was compared to that of LPS and TLR3 agonist poly(I:C) using a human skin explant model with intradermal injections for the administration of the agonists. Intradermal injection of GLA-SE or LPS, but not GLA-AF, enhanced the emigration of CD1a(high)/langerin(+) Langerhans cells (LCs), but not dermal DCs (DDCs). LCs and CD14(-) DDCs exhibited an enhanced mature phenotype following intradermal administration of either of the two GLA formulations tested, similar to DCs that emigrated from LPS-injected skin. However, only injection of GLA-SE resulted in a significant increase in the production of the wide range of cytokines that is observed with LPS. Moreover, DCs that emigrated from GLA-SE-injected skin induced stronger CD4(+) T-cell activation, as indicated by a more pronounced T-cell proliferation, than DCs from skin injected with GLA-AF or LPS. Altogether, our data show that GLA-SE has a notable potency to stimulate the function of skin DCs, indicating that GLA-SE may be a good candidate as adjuvant for vaccines administered via the intradermal route.     

Early origins of polycystic ovary syndrome

The prenatally androgenised female rhesus monkey has become a model for polycystic ovary syndrome (PCOS) in women, with early prenatal androgenisation entraining a permanent PCOS-like phenotype characterised by luteinising hormone (LH) hypersecretion due to reduced hypothalamic sensitivity to steroid negative feedback and relative insulin excess associated with increased abdominal adiposity. These combined reproductive and metabolic abnormalities occur in combination with ovarian hyperandrogenism and follicular arrest in adulthood, and with premature follicle differentiation and impaired embryo development during gonadotrophin therapy for in vitro fertilization (IVF). The ability of prenatal androgen excess in fetal rhesus monkeys to entrain multiple organ systems in utero provides evidence that the hormonal environment of intrauterine life programmes target tissue differentiation, raising the possibility that hyperandrogenism in human fetal development promotes PCOS in adulthood. This hypothesis developed in prenatally androgenised female rhesus monkeys, however, also must include data from clinical studies of PCOS to clarify the homology between human and non-human primates in intrafollicular steroidogenesis and its impact on oocyte developmental competency. By doing so, future studies promise to develop new clinical strategies that will lead to improved pregnancy outcome and reduced pregnancy loss in women with disorders of insulin action, including PCOS, obesity and diabetes mellitus.     

Prevalence of Balantidium coli Infection in Bred Rhesus Monkeys (Macaca mulatta) in Guangxi,southern China

Background

Balantidium coli infects humans, primates and pigs, causing serious diarrhea and dysentery. Little information on the prevalence of B. coli in primates is available in China. This investigation was conducted to determine the prevalence of B. coli infection in bred rhesus monkeys in Guangxi Zhuang Nationality Autonomous Region (GZNAR), southern China.

Methods

A total of 120 fecal samples were collected from rhesus monkeys bred in cages in GZNAR and B. coli cysts and/or trophozoites were examined microscopically after sedimentation with water in May 2013.

Results

(64.2%) samples were tested positive. The prevalence was 65% (39/60) and 63.3% (38/60) in female and male monkeys, respectively. 80% (48/60) cages in this nonhuman primate center were positive for B. coli.

Conclusion

The present survey revealed high circulation of B. coli in bred rhesus monkeys in GZNAR, which poses potential threats to animal and human health.