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.

Fig 1

Recombinant AnAPN1, produced in E. coli, exhibits intrinsic antigenicity in mice and is capable of eliciting functional malaria transmission-blocking antibodies in the absence of adjuvant. (a and b) A single-prime and three-boost immunization dose regimen (2 μg/dose at 2-week intervals) produces a robust antigen-specific antibody response in mice compared to that seen in controls (mice receiving only incomplete Freund's adjuvant). Sera have been pooled from 4 or 5 mice/cohort. (c and d) A single prime and -boost dose at day 28 (2 μg/dose) produce an antibody response for week 8 postimmunization sera comparable to that seen in panel b. Sera were pooled from 4 or 5 mice/cohort. (e and f) Direct feeding assay (DFA) results demonstrated that mice receiving AnAPN1 (2 μg/dose) in the absence of adjuvant elicited transmission-blocking antibodies against Plasmodium berghei in Anopheles stephensi mosquitoes. Control group values are shown as open circles (corresponding to panel c). Treatment group values are shown as filled circles (corresponding to panel d). Horizontal bars indicate median oocyst numbers per mosquito midgut. The lower table highlights the statistical significance of the Pr value of 0.031430 as determined by the GEE model. Data are representative of two independent biological replicate cohort immunization studies.

Fig 2

Immunization of mice with AnAPN1-Alhydrogel elicited a potent antigen-specific humoral response and functional, malaria parasite transmission-blocking antibodies. Sera were collected from the satellite mouse control cohort C (a) and APN cohort D (b). Only sera from cohort D demonstrated an AnAPN1-specific antibody response. Mice were immunized following the prime–three-boost schedule (5 μg/dose) used for mouse cohorts A and B, which were subsequently analyzed for gross and subpatent histopathologies. (c) Representative results from passive immunization feeding assays (PIFA) that demonstrate the potent transmission-blocking activity of AnAPN1-specific IgG (100 μg/ml) collected from mice in cohorts B and D against Plasmodium berghei in Anopheles stephensi mosquitoes. We observed reductions in median oocyst infection intensity between 80 to 100%. Each set of pre- and postexperiment values represents a single biological replicate. *, P < 0.05; **, P < 0.01.

Fig 3

An immunohistochemical study of normal human tissue sections with rabbit anti-AnAPN1 IgG and a complete histopathological analysis of mice following AnAPN1-Alhydrogel immunizations demonstrated the complete absence of cross-specific staining of human APN homologs and gross histopathologies in mice. (a) Frozen sections were stained with anti-CD13, MAb (WM15 clone, positive control), and polyclonal rabbit anti-AnAPN1 IgG and detected with anti-mouse Texas Red and anti-rabbit Alexa Fluor 488-conjugated antibody. Images were acquired at 200× and 400× magnifications. Differential interference contrast (DIC) images are provided for orientation at 200×, and the inset corresponds to the anti-CD13 and anti-AnAPN1 staining and the merged image, which were all acquired at 400×. DAPI (4′,6-diamidino-2-phenylindole) was used to stain nuclei and appear blue. The signal observed in the 460- to 490-nm band pass filter range across all AnAPN1 images corresponds to the marked autofluorescence observed for these tissues. (b) A summary of the acute (days 1 to 14) and chronic (90 days) histopathology results in control (Alhydrogel) and treatment (AnAPN1-Alhydrogel) mouse cohorts A and B (see the Fig. 2 legend), respectively. SHL, small hepatic lesions; LNE, lymph node enlargements; UE, urinary tract edema; ME, mild enteritis.

Fig 4

AnAPN1 mouse potency study. (a) Anti-AnAPN1 IgG on day 28 post-prime vaccination. Sera were diluted at 1:6,000. Note that mice vaccinated with 50 μg of AnAPN1 alone generated an anti-AnAPN1 IgG response corroborating the data in Fig. 1. However, a statistically significant (P = 0.0237) increase in anti-AnAPN1 IgG (AU) on day 42 post-prime vaccination (day 14 postboost) was found on addition of 400 μg of Alhydrogel to 50 μg of AnAPN1. The reactivity threshold is the concentration of antibody corresponding to the interpolated intersection of the lower asymptote of the upper 95% confidence limit with the lower 95% confidence limit of the standard data. (b) Anti-AnAPN1 IgG on day 42 post-prime vaccination (14 days postboost). Sera were diluted at 1:6,000. (c) Immunoglobulin isotype subclassification of antisera from each mouse that received the 50-μg dose of AnAPN1 formulated with or without Alhydrogel.

Fig 5

Potency of AnAPN1 as demonstrated by the median 50% effective dose (ED₅₀) and lymphoproliferative responses. (a) Proc Probit (SAS 9.2) modeling of the dose-response curve using sera collected on day 28 (4 weeks after prime vaccination of BALB/c mice) indicates an ED₅₀ in the presence of adjuvant of 21.25 μg (95% fiducial limits of 16.37 μg and 28.28 μg). (b) Immunization with unformulated and formulated AnAPN1 induces cellular proliferation in spleen cells of BALB/c mice. Splenic lymphocytes harvested from BALB/c mice 42 days after primary immunization and (14 days after boost) with various doses of AnAPN1 formulated with Alhydrogel (groups 1 to 3 [G1 to G3]). Lymphoproliferation is expressed as the stimulation index (SI) with levels of bromodeoxyuridine (BrdU) in cells cultured with concanavalin A (Con A) or AnAPN1 (17.5 and 35 μg/ml) over the levels in cells in control wells (no antigen) after 5 days.