Brian Keegan

Vaccination of Monkeys with Recombinant Plasmodium falciparum Apical Membrane Antigen 1 Confers Protection against Blood-Stage Malaria

ABSTRACT A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy. We evaluated the efficacy of a form of P. falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P. falciparum parasites. Significant protection from this otherwise lethal challenge with P. falciparum was observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized. The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial. The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.

An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection

Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR‐1. Ac‐APR‐1 from the dog hookworm, Ancylostoma caninum, protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood‐feeding. Toward developing a human hookworm vaccine, we expressed both wild‐type (Na‐APR‐1wt) and mutant (Na‐APR‐1mut—mutagenesis of the catalytic aspartic acids) forms of Na‐APR‐1 from the human hookworm, Necator americanus. Refolded Na‐APR‐1wt was catalytically active, and Na‐APR‐1mut was catalytically inactive but still bound substrates. Vaccination of canines with Na‐APR‐1mut and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens (P=0.034) and weight loss (P=0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Na‐APR‐1mut induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na‐APR‐1wt and APR‐1 orthologues from three other hookworm species that infect humans. IgG1 against Na‐APR‐1mut was the most prominently detected antibody in sera from people resident in high‐transmission areas for N. americanus, indicating that natural boosting may occur in exposed humans. Na‐APR‐1mut is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease.—Pearson, M. S., Bethony, J. M., Pickering, D. A., de Oliveira, L. M., Jariwala, A., Santiago, H., Miles, A. P., Zhan, B., Jiang, D., Ranjit, N., Mulvenna, J., Tribolet, L., Plieskatt, J., Smith, T., Bottazzi, M. E., Jones, K., Keegan, B., Hotez, P. J., Loukas, A. An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection. FASEB J. 23, 3007–3019 (2009). www.fasebj.org

Expression, immunogenicity, histopathology, and potency of a mosquito-based malaria transmission-blocking recombinant vaccine

ABSTRACT Vaccines have been at the forefront of global research efforts to combat malaria, yet despite several vaccine candidates, this goal has yet to be realized. A potentially effective approach to disrupting the spread of malaria is the use of transmission-blocking vaccines (TBV), which prevent the development of malarial parasites within their mosquito vector, thereby abrogating the cascade of secondary infections in humans. Since malaria is transmitted to human hosts by the bite of an obligate insect vector, mosquito species in the genus Anopheles, targeting mosquito midgut antigens that serve as ligands for Plasmodium parasites represents a promising approach to breaking the transmission cycle. The midgut-specific anopheline alanyl aminopeptidase N (AnAPN1) is highly conserved across Anopheles vectors and is a putative ligand for Plasmodium ookinete invasion. We have developed a scalable, high-yield Escherichia coli expression and purification platform for the recombinant AnAPN1 TBV antigen and report on its marked vaccine potency and immunogenicity, its capacity for eliciting transmission-blocking antibodies, and its apparent lack of immunization-associated histopathologies in a small-animal model.

Advancing a multivalent ‘Pan-anthelmintic’ vaccine against soil-transmitted nematode infections

The Sabin Vaccine Institute Product Development Partnership is developing a Pan-anthelmintic vaccine that simultaneously targets the major soil-transmitted nematode infections, in other words, ascariasis, trichuriasis and hookworm infection. The approach builds off the current bivalent Human Hookworm Vaccine now in clinical development and would ultimately add both a larval Ascaris lumbricoides antigen and an adult-stage Trichuris trichiura antigen from the parasite stichosome. Each selected antigen would partially reproduce the protective immunity afforded by UV-attenuated Ascaris eggs and Trichuris stichosome extracts, respectively. Final antigen selection will apply a ranking system that includes the evaluation of expression yields and solubility, feasibility of process development and the absence of circulating antigen-specific IgE among populations living in helminth-endemic regions. Here we describe a five year roadmap for the antigen discovery, feasibility and antigen selection, which will ultimately lead to the scale-up expression, process development, manufacture, good laboratory practices toxicology and preclinical evaluation, ultimately leading to Phase 1 clinical testing.

Time course study of the antigen-specific immune response to a PLGA microparticle vaccine formulation.

Microparticle-based vaccine delivery systems are known to promote enhanced immune responses to protein antigens and can elicit TH1-biased responses when used in combination with Toll-like receptor (TLR) agonists. It is important to understand the kinetics of the immune responses to microparticle-based protein vaccines in order to predict the duration of protective immunity and to optimize prime-boost vaccination regimens. We carried out a 10-week time course study to investigate the magnitude and kinetics of the antibody and cellular immune responses to poly(lactic-co-glycolic acid) (PLGA) microparticles containing 40 μg ovalbumin (OVA) protein and 16 μg CpG-ODN adjuvant (MP/OVA/CpG) in comparison to OVA-containing microparticles, soluble OVA plus CpG, or OVA formulated with Alhydrogel(®) aluminum adjuvant. Mice vaccinated with MP/OVA/CpG developed the highest TH1-associated IgG2b and IgG2c antibody titers, while also eliciting TH2-associated IgG1 antibody titers on par with Alhydrogel(®)-formulated OVA, with all IgG subtype titers peaking at day 56. The MP/OVA/CpG vaccine also induced the highest antigen-specific splenocyte IFN-γ responses, with high levels of IFN-γ responses persisting until day 42. Thus the MP/OVA/CpG formulation produced a sustained and heightened humoral and cellular immune response, with an overall TH1 bias, while maintaining high levels of IgG1 antibody equivalent to that seen with Alhydrogel(®) adjuvant. The time course kinetics study provides a useful baseline for designing vaccination regimens for microparticle-based protein vaccines.

Mechanistic and Single-Dose In Vivo Therapeutic Studies of Cry5B Anthelmintic Action against Hookworms

Background Hookworm infections are one of the most important parasitic infections of humans worldwide, considered by some second only to malaria in associated disease burden. Single-dose mass drug administration for soil-transmitted helminths, including hookworms, relies primarily on albendazole, which has variable efficacy. New and better hookworm therapies are urgently needed. Bacillus thuringiensis crystal protein Cry5B has potential as a novel anthelmintic and has been extensively studied in the roundworm Caenorhabditis elegans. Here, we ask whether single-dose Cry5B can provide therapy against a hookworm infection and whether C. elegans mechanism-of-action studies are relevant to hookworms. Methodology/Principal Findings To test whether the C. elegans invertebrate-specific glycolipid receptor for Cry5B is relevant in hookworms, we fed Ancylostoma ceylanicum hookworm adults Cry5B with and without galactose, an inhibitor of Cry5B-C. elegans glycolipid interactions. As with C. elegans, galactose inhibits Cry5B toxicity in A. ceylanicum. Furthermore, p38 mitogen-activated protein kinase (MAPK), which controls one of the most important Cry5B signal transduction responses in C. elegans, is functionally operational in hookworms. A. ceylanicum hookworms treated with Cry5B up-regulate p38 MAPK and knock down of p38 MAPK activity in hookworms results in hypersensitivity of A. ceylanicum adults to Cry5B attack. Single-dose Cry5B is able to reduce by >90% A. ceylanicum hookworm burdens from infected hamsters, in the process eliminating hookworm egg shedding in feces and protecting infected hamsters from blood loss. Anthelmintic activity is increased about 3-fold, eliminating >97% of the parasites with a single 3 mg dose (∼30 mg/kg), by incorporating a simple formulation to help prevent digestion in the acidic stomach of the host mammal. Conclusions/Significance These studies advance the development of Cry5B protein as a potent, safe single-dose anthelmintic for hookworm therapy and make available the information of how Cry5B functions in C. elegans in order to study and improve Cry5B function against hookworms.

Potency testing for the experimental Na-GST-1 hookworm vaccine

Over the next decade, a new generation of vaccines will target the neglected tropical diseases (NTDs). The goal of most NTD vaccines will be to reduce the morbidity and decrease the chronic debilitating nature of these often-forgotten infections – outcomes that are hard to measure in the traditional potency testing paradigm. The absence of measurable correlates of protection, a lack of permissive animal models for lethal infection, and a lack of clinical indications that do not include the induction of sterilizing immunity required us to reconsider the traditional bioassay methods for determining vaccine potency. Owing to these limitations, potency assay design for NTD vaccines will increasingly rely on a paradigm where potency testing is one among many tools to ensure that a manufacturing process yields a product of consistent quality. Herein, we discuss the evolution of our thinking regarding the design of a potency assay along these newly defined lines and its application to the release of the experimental Necator americanus-glutathione-S- transferase-1 (Na-GST-1) vaccine to prevent human hookworm infection. We discuss the necessary steps to accomplish the design and implementation of such a new potency assay as a resource for the burgeoning NTD vaccine community. Our experience is that much of the existing information is proprietary and needs to be pulled together in a single source to aid in our overall understanding of potency testing.

Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease

ABSTRACT A therapeutic vaccine for human Chagas disease is under development by the Sabin Vaccine Institute Product Development Partnership. The aim of the vaccine is to significantly reduce the parasite burden of Trypanosoma cruzi in humans, either as a standalone product or in combination with conventional chemotherapy. Vaccination of mice with Tc24 formulated with monophosphoryl-lipid A (MPLA) adjuvant results in a Th1 skewed immune response with elevated IgG2a and IFNγ levels and a statistically significant decrease in parasitemia following T. cruzi challenge. Tc24 was therefore selected for scale-up and further evaluation. During scale up and downstream process development, significant protein aggregation was observed due to intermolecular disulfide bond formation. To prevent protein aggregation, cysteine codons were replaced with serine codons which resulted in the production of a non-aggregated and soluble recombinant protein, Tc24-C4. No changes to the secondary structure of the modified molecule were detected by circular dichroism. Immunization of mice with wild-type Tc24 or Tc24-C4, formulated with E6020 (TLR4 agonist analog to MPLA) emulsified in a squalene-oil-in-water emulsion, resulted in IgG2a and antigen specific IFNγ production levels from splenocytes that were not significantly different, indicating that eliminating putative intermolecular disulfide bonds had no significant impact on the immunogenicity of the molecule. In addition, vaccination with either formulated wild type Tc24 or Tc24-C4 antigen also significantly increased survival and reduced cardiac parasite burden in mice. Investigations are now underway to examine the efficacy of Tc24-C4 formulated with other adjuvants to reduce parasite burden and increase survival in pre-clinical studies.

Fusion of Na‐ASP‐2 with human immunoglobulin Fcγ abrogates histamine release from basophils sensitized with anti‐Na‐ASP‐2 IgE

Na‐ASP‐2 is a major protein secreted by infective third‐stage larvae (L3) of the human hookworm Necator americanus upon host entry. It was chosen as a lead vaccine candidate for its ability to elicit protective immune responses. However, clinical development of this antigen as a recombinant vaccine was halted because it caused allergic reactions among some of human volunteers previously infected with N. americanus. To prevent IgE‐mediated allergic reactions induced by Na‐ASP‐2 but keep its immunogenicity as a vaccine antigen, we designed and tested a genetically engineered fusion protein, Fcγ/Na‐ASP‐2, composed of full‐length Na‐ASP‐2 and truncated human IgG Fcγ1 that targets the negative signalling receptor FcγRIIb expressed on pro‐allergic cells. The chimeric recombinant Fcγ/Na‐ASP‐2 protein was expressed in Pichia pastoris and shared the similar antigenicity as native Na‐ASP‐2. Compared to Na‐ASP‐2, the chimeric fusion protein efficiently reduced the release of histamine in human basophils sensitized with anti‐Na‐ASP‐2 IgE obtained from individuals living in a hookworm‐endemic area. In dogs infected with canine hookworm, Fcγ/Na‐ASP‐2 resulted in significantly reduced immediate‐type skin reactivity when injected intradermally compared with Na‐ASP‐2. Hamsters vaccinated with Fcγ/Na‐ASP‐2 formulated with Alhydrogel® produced specific IgG that recognized Na‐ASP‐2 and elicited similar protection level against N. americanus L3 challenge as native Na‐ASP‐2.

The metastasis‐associated protein‐1 gene encodes a host permissive factor for schistosomiasis, a leading global cause of inflammation and cancer

Schistosoma haematobium is responsible for two‐thirds of the worlds 200 million to 400 million cases of human schistosomiasis. It is a group 1 carcinogen and a leading cause of bladder cancer that occurs after years of chronic inflammation, fibrosis, and hyperproliferation in the host liver. The coevolution of blood flukes of the genus Schistosoma and their human hosts is paradigmatic of long‐term parasite development, survival, and maintenance in mammals. However, the contribution of host genes, especially those discrete from the immune system, necessary for parasite establishment and development remains poorly understood. This study investigated the role of metastasis‐associated protein‐1 gene (Mta1) product in the survival of S. haematobium and productive infection in the host. Using a Mta−1 null mouse model, here we provide genetic evidence to suggest that MTA1 expression positively influences survival and/or maturation of schistosomes in the host to patency, as we reproducibly recovered significantly fewer S. haematobium worms and eggs from Mta1−/− mice than wild−type mice. In addition, we found a distinct loss of cytokine interdependence and aberrant Th1 and Th2 cytokine responses in the Mta1−/− mice compared to age‐matched wild‐type mice. Thus, utilizing this Mta1‐null mouse model, we identified a distinct contribution of the mammalian MTA1 in establishing a productive host–parasite interaction and thus revealed a host factor critical for the optimal survival of schistosomes and successful parasitism. Moreover, MTA1 appears to play a significant role in driving inflammatory responses to schistosome egg–induced hepatic granulomata reactions, and thus offers a survival cue for parasitism as well as an obligatory contribution of liver in schistosomiasis. Conclusion: These findings raise the possibility to develop intervention strategies targeting MTA1 to reduce the global burden of schistosomiasis, inflammation, and neoplasia. (HEPATOLOGY 2011;)