Roman F. Wolf

Protective and antifecundity effects of Sm-p80 based DNA vaccine formulation against Schistosoma mansoni in a nonhuman primate model

Schistosomiasis is an important parasitic disease for which there is no available vaccine. We have focused on a functionally important antigen of Schistosoma mansoni, Sm-p80, as a vaccine candidate because of its consistent immunogenicity, protective potential and antifecundity effect observed in murine models; and for its pivotal role in the immune evasion process. In the present study we report that an Sm-p80-based DNA vaccine formulation confers 38% reduction in worm burden in a nonhuman primate model, the baboon (Papio anubis). Animals immunized with Sm-p80-pcDNA3 exhibited a decrease in egg production by 32%. Sm-p80 DNA elicited specific immune responses that include IgG; its subtypes IgG1 and IgG2; and IgM in vaccinated animals. Peripheral blood mononuclear cells (PBMCs) from immunized animals when stimulated in vitro with Sm-p80 produced appreciably more Th1 response enhancing cytokines (IL-2, IFN-gamma) than Th2 response enhancing cytokines (IL-4, IL-10). PBMCs produced appreciably more spot-forming units for INF-gamma than for IL-4 in enzyme-linked immunosorbent spot (ELISPOT) assays. Overall it appears that even though a mixed (Th1/Th2) type of humoral antibody response was generated following immunization with Sm-p80; the dominant protective immune response is Th1 type. These data reinforce the potential of Sm-p80 as an excellent vaccine candidate for schistosomiasis.

Late onset of development of natural anti‐nonGal antibodies in infant humans and baboons: implications for xenotransplantation in infants

If an ABO‐incompatible heart is transplanted into an infant before natural antibodies have developed to the specific donor carbohydrate A/B antigen(s), then B‐cell tolerance to the donor A/B antigen is achieved, and these antibodies never develop. Anti‐carbohydrate antibodies play a role in the rejection of wild type (WT) and α1,3‐galactosyltransferase gene‐knockout (GT‐KO) pig xenografts. We investigated development of these antibodies in infant baboons and humans. Serum samples from infant baboons (n = 42) and humans (n = 42) were tested by flow cytometry for immunoglobulin M and immunoglobulin G binding to peripheral blood mononuclear cells from WT and GT‐KO pigs, and for complement‐dependent cytotoxicity. The presence of anti‐blood group antibodies was tested in baboon serum. In infant baboons and humans, cytotoxic anti‐Galα1,3Gal antibodies develop during the first 3 months, and steadily increase with age, whereas cytotoxic anti‐nonGal antibodies are either absent or minimal in the majority of cases throughout the first year of life. Anti‐blood group antibodies were not detected before 16 weeks of age. Our data suggest GT‐KO pig organ/cell transplants could be carried out in early infancy in the absence of preformed cytotoxic anti‐nonGalα1,3Gal antibodies.

Maternal and Neonatal Vaccination Protects Newborn Baboons From Pertussis Infection

BACKGROUND The United States is experiencing a pertussis resurgence that resulted in a 60-year high of 48 000 cases in 2012. The majority of hospitalizations and deaths occur in infants too young to be vaccinated. Neonatal and maternal vaccination have been proposed to protect newborns until the first vaccination, currently recommended at 2 months of age. These interventions result in elevated anti-Bordetella pertussis titers, but there have been no studies demonstrating that these measures confer protection. METHODS Baboons were vaccinated with acellular pertussis vaccine at 2 days of age or at 2 and 28 days of age. To model maternal vaccination, adult female baboons primed with acellular pertussis vaccine were boosted in the third trimester of pregnancy. Neonatally vaccinated infants, infants born to vaccinated mothers, and naive infants born to unvaccinated mothers were infected with B. pertussis at 5 weeks of age. RESULTS Naive infant baboons developed severe disease when challenged with B. pertussis at 5 weeks of age. Baboons receiving acellular pertussis vaccine and infants born to mothers vaccinated at the beginning of their third trimester were protected. CONCLUSIONS Our results demonstrate that neonatal vaccination and maternal vaccination confer protection in the baboon model and support further study of these strategies for protection of newborns from pertussis.

Induced antitumor immunity against DMBA‐4 metastatic mammary tumors in rats using laser immunotherapy

Induced antitumor immunity is a highly effective and long‐term cure for cancer, particularly for metastatic tumors. Laser immunotherapy was developed to induce such an immunologic response. It involves intratumoral administration of a light‐absorbing dye and a specially formulated immunoadjuvant, followed by noninvasive irradiation of a near‐infrared laser. Treatment of DMBA‐4 metastatic mammary tumors in rats with this approach has resulted in local control of primary tumors and eradication of untreated distant metastases. After laser immunotherapy, rats were resistant to tumor rechallenge and developed immunity, which could be adoptively transferred. To better understand the immunity induced in this tumor model, immunization using freeze–thaw DMBA‐4 cell lysates was performed, followed by tumor challenge 21 days later. Tumor cell lysate immunization delayed the emergence of metastases but did not provide immunity against the tumor challenge. Also performed was surgical resection of primary tumors before the observation of metastatic tumors. Removal of primary tumors was unsuccessful at changing the course of tumor progression. Tumors re‐emerged at the primary sites, and metastases developed at multiple remote sites. In contrast, tumor‐bearing rats successfully treated by laser immunotherapy experienced tumor regression and eradication and developed strong resistance to repeated challenges by tumor cells of the same type. Our results show that laser immunotherapy could have potential for the treatment of metastatic tumors by inducing tumor‐specific, long‐lasting immunity.

Cross-species protection: Schistosoma mansoni Sm-p80 vaccine confers protection against Schistosoma haematobium in hamsters and baboons

The ability of the Schistosoma mansoni antigen, Sm-p80, to provide cross-species protection against Schistosoma haematobium challenge was evaluated in hamster and baboon models. Pronounced reduction in worm burden (48%) and in tissue egg load (64%) was observed in hamsters vaccinated with recombinant Sm-p80 admixed with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Similarly, in baboons, the Sm-p80/GLA-SE vaccine produced a 25% reduction in S. haematobium adult worms and decreased the egg load in the urinary bladder by 64%. A 40% and 53% reduction in fecal and urine egg output, respectively, was observed in vaccinated baboons. A balanced pro-inflammatory (Th17 and Th1) and Th2 type of response was generated after vaccination and appears indicative of augmented prophylactic efficacy. These data on cross-species protection coupled with the prophylactic, therapeutic and antifecundity efficacy against the homologous parasite, S. mansoni, reinforces Sm-p80 as a promising vaccine candidate. It is currently being prepared for GMP-compliant manufacture and for further pre-clinical development leading to human clinical trials. These results solidify the expectation that the Sm-p80 vaccine will provide relief for both the intestinal and the urinary schistosomiasis and thus will be greatly beneficial in reducing the overall burden of schistosomiasis.

Sm-p80—Based DNA Vaccine Provides Baboons with Levels of Protection against Schistosoma mansoni Infection Comparable to Those Achieved by the Irradiated Cercarial Vaccine

To date, no vaccine is available to prevent human schistosomiasis. We have targeted a protein of Schistosoma mansoni that plays an important role in the surface membrane renewal process, a mechanism widely believed to be utilized by the parasite as an immune evasion strategy. Sm-p80 antigen is a promising vaccine target because of its documented immunogenicity, protective efficacy, and antifecundity effects observed in both experimental murine and nonhuman primate models of this infectious disease. In the present study, we report that, in a vector approved for human use (VR1020), an Sm-p80-based DNA vaccine formulation confers a 46% reduction in the worm burden in a baboon (Papio anubis) model. Baboons vaccinated with Sm-p80-VR1020 had a 28% decrease in egg production after challenge with the infectious parasite. Sm-p80-VR1020 vaccine elicited robust immune responses to specific antigen Sm-p80, including immunoglobulin (Ig) G, its subtypes IgG1 and IgG2, and IgA and IgM in vaccinated animals. When stimulated in vitro with recombinant Sm-p80, peripheral blood mononuclear cells and splenocytes from baboons vaccinated with Sm-p80-VR1020 produced considerably higher levels of T helper 1 response-enhancing cytokines (interleukin [IL]-2 and interferon-gamma) than T helper 2 (Th2) response-enhancing cytokines (IL-4 and IL-10). Peripheral blood mononuclear cells produced a significantly higher number of spot-forming units for interferon-gamma than for IL-4 in enzyme-linked immunosorbent spot assays. A mixed T helper 1/T helper 2 type of humoral and T cell responses was generated after immunization with Sm-p80-VR1020. These findings again highlight the potential of Sm-p80 as a promising vaccine candidate for schistosomiasis.

Complex Role of Hemoglobin and Hemoglobin-Haptoglobin Binding Proteins in Haemophilus influenzae Virulence in the Infant Rat Model of Invasive Infection

ABSTRACT Haemophilus influenzae requires an exogenous heme source for aerobic growth in vitro. Hemoglobin or hemoglobin-haptoglobin satisfies this requirement. Heme acquisition from hemoglobin-haptoglobin is mediated by proteins encoded by hgp genes. Both Hgps and additional proteins, including those encoded by the hxu operon, provide independent pathways for hemoglobin utilization. Recently we showed that deletion of the set of three hgp genes from a nontypeable strain (86-028NP) of H. influenzae attenuated virulence in the chinchilla otitis media model of noninvasive disease. The present study was undertaken to investigate the role of the hgp genes in virulence of the wild-type serotype b clinical isolate HI689 in the infant rat model of hematogenous meningitis, an established model of invasive disease requiring aerobic growth. Bacteremia of high titer and long duration (>14 days) and histopathologically confirmed meningitis occurred in >95% of infant rats challenged at 5 days of age with strain HI689. While mutations disrupting either the Hgp- or Hxu-mediated pathway of heme acquisition had no effect on virulence in infant rats, an isogenic mutant deficient for both pathways was unable to sustain bacteremia or produce meningitis. In contrast, mutations disrupting either pathway decreased the limited ability of H. influenzae to initiate and sustain bacteremia in weanling rats. Biochemical and growth studies also indicated that infant rat plasma contains multiple heme sources that change with age. Taken together, these data indicate that both the hgp genes and the hxuC gene are virulence determinants in the rat model of human invasive disease.

Use of an Sm-p80–Based Therapeutic Vaccine to Kill Established Adult Schistosome Parasites in Chronically Infected Baboons

No vaccines are available for human use for any parasitic infections, including the helminthic disease schistosomiasis. Sm-p80, the large subunit of Schistosoma mansoni calpain, is a leading antigen candidate for a schistosomiasis vaccine. Prophylactic and antifecundity efficacies of Sm-p80 have been tested using a variety of vaccine approaches in both rodent and nonhuman primate models. However, the therapeutic efficacy of a Sm-p80-based vaccine had not been determined. In this study, we evaluated the therapeutic efficacy of Sm-p80 by using 2 different strategies and 3 Sm-p80-based vaccine formulations in baboons. Vaccine formulations were able to decrease established adult worms by 10%-36%, reduce retention of eggs in tissues by 10%-57%, and decrease egg excretion in feces by 13%-33%, compared with control formulations. Marked differences were observed in B and T cell immune correlates between vaccinated and control animals. This is the first report of killing of established adult schistosome worms by a vaccine. In addition to distinct prophylactic efficacy of Sm-p80, this study adds to the evidence that Sm-p80 is a potentially important antigen with both substantial prophylactic and therapeutic efficacies. These data reinforce that Sm-p80 should be moved forward along the path toward human clinical trials.

Infant baboons infected with respiratory syncytial virus develop clinical and pathological changes that parallel those of human infants.

Respiratory syncytial virus (RSV) infection of the lower respiratory tract is the leading cause of respiratory failure among infants in the United States of America and annually results in >300,000 deaths worldwide. Despite the importance of RSV, there is no licensed vaccine, and no specific form of therapy. This is largely due to the absence of an appropriate animal model for the evaluation of vaccines and therapeutic agents. We inoculated anesthetized infant (4 wk) baboons (Papio anubis) with a human strain of RSV intranasally or intratracheally. Baboons were monitored daily for clinical changes. Anesthetized baboons were intubated at various intervals, and bronchoalveolar lavage (BAL) was performed for viral culture and determination of leukocyte counts. Sham-infected baboons served as controls. Necropsies were performed on infected baboons on days 1, 3, 5, 8, or 13 after inoculation, with pathological analysis and immunohistochemical staining of lung tissues to detect RSV antigen. Infected baboons developed tachypnea and reduced oxygenation peaking from 4 to 8 days after infection and persisting for ≥14 days. Virus was recoverable in BAL fluid up to 8 days following infection. Necropsy revealed intense interstitial pneumonia, sloughing of the bronchiolar epithelium, and obstruction of the bronchiolar lumen with inflammatory cells and sloughed epithelial cells. RSV antigen was identified in bronchiolar and alveolar epithelium. We conclude that RSV-infected infant baboons develop clinical and pathological changes that parallel those observed in human infants with RSV infection. The infant baboon represents a much-needed model for studying the pathogenesis of RSV infection and evaluating antivirals and vaccines.

Age-Associated Alteration in Innate Immune Response in Captive Baboons

Baboons are an ideal model for studies of human inflammatory response due to their physiological and immunological similarities to people; however; little is known about how age affects immune function in the baboon. We sought to determine if baboons show age-related innate immune changes similar to that described in people. Age was correlated with increased serum C-reactive protein and interleukin-6 or, however, no change in interleukin-10 concentration was observed (n = 120 baboons). Cytokine release from unstimulated peripheral blood mononuclear cells as well as following immune (lipopolysaccharide) stimulation increased with age. When whole blood was assayed, both lipopolysaccharide stimulated and unstimulated samples showed an age-related increase in interleukin-6 response, although the unstimulated cytokine response was reduced compared with that observed in peripheral blood mononuclear cells. Tumor necrosis factor-α response was not related to age. Cytokine response in lipopolysaccharide-stimulated whole blood was negatively correlated with serum DHEA-S concentration and positively correlated with TGF-β concentration.