Chronic helminth infection does not impair immune response to malaria transmission blocking vaccine Pfs230D1-EPA/Alhydrogel® in mice

Abstract

Introduction: Malaria transmission blocking vaccines (TBV) are innovative approaches that aim to induce immunity in humans against Plasmodium during mosquito stage, neutralizing the capacity of the infected vectors to transmit malaria. Pfs230D1-EPA/Alhydrogel®, a promising protein-protein conjugate malaria TBV, is currently being tested in human clinical trials in areas where P. falciparum malaria is coendemic with helminth parasites. Helminths are complex metazoans that share the master capacity to downregulate the host immune response towards themselves and also to bystander antigens, including vaccines. However, it is not known whether the activity of a protein-based malaria TBV may be affected by a chronic helminth infection.

Methods: Using an experimental murine model for a chronic helminth infection (Heligmosomoides polygyrus bakeri - Hpb), we evaluated whether prior infection alters the activity of Pfs230D1-EPA/Alhydrogel® TBV in mice.

Results: After establishment of a chronic infection, characterized by a marked increase of parasite antigen-specific IgG1, IgA and IgE antibody responses, concomitant with an increase of systemic IL-10, IL-5 and IL-6 levels, the Hpb-infected mice were immunized with Pfs230D1-EPA/Alhydrogel® and the vaccine-specific immune response was compared with that in non-infected immunized mice. TBV immunizations induced an elevated vaccine specific-antibody response, however Pfs230D1 specific-IgG levels were similar between infected and uninfected mice at days 15, 25 and 35 post-vaccination. Absolute numbers of Pfs230D1-activated B cells generated in response to the vaccine were also similar among the vaccinated groups. Finally, vaccine activity assessed by reduction of oocyst number in P. falciparum infected mosquitoes was similar between Hpb-infected and immunized mice with non-infected immunized mice.

Conclusion: Pfs230D1-EPA/Alhydrogel® efficacy is not impaired by a chronic helminth infection in mice.

Keywords: H. polygyrus bakeri; Helminth intestinal infection; Immunoregulation; Malaria transmission blocking vaccine; P. falciparum; Pfs230.

Fig. 1

Immunization strategy for Pfs230D1-EPA/Alhydrogel® in mice previously infected (or not infected) with H. polygyrus bakeri. After 28 days of infection with 200 infective Larvae (L3i) of H. polygyrus bakeri, BALB/c mice (n = 10 for each group) were vaccinated intramuscularly with two doses of 1 µg of Pfs230D1-EPA. Blood was collected at days −1, 15, 25 and 35.

Fig. 2

Hpb Infection pattern was sustained after immunization. (a) Amount of Hpb eggs were similar in stools collected in animals infected only or both infected/vaccinated at days −3 (p = 0.574), 25 (p = 0.841) and 35 (p = 0.999). (b) ELISA optical density in response to Hpb antigen in sera. Sera collected at days −1, 15, 25 and 35 were used to assess IgG1, IgG2a, IgG3, IgE and IgA responses to H. polygyrus bakeri. Data are shown as mean + SEM.

Fig. 3

Pfs230D1 IgG titers in sera from mice. Mice were previously infected or not with H. polygyrus baker, and 28 days immunized or not with Pfs230D1-EPA/Alhydrogel® (n = 10 for each of the 4 groups, combined from two different immunization studies). Pfs230D1-specific antibodies generated with 1 µg of Pfs230D1 vaccine injected intramuscularly were measured by ELISA. Data are shown as mean + SEM.

Fig. 4

Serum cytokines from mice immunized with Pfs230D1-EPA/Alhydrogel® and previously infected with H. polygyrus bakeri (n = 10 for each group, determined in two different experiments). IL-10, TNF-a, IL-5, IL-13, IL-6 and IFN-y levels in sera were quantified by Luminex assay. Data are shown as mean + SEM.

Supplementary Fig. 1

Fig. 5

Pfs230D1-specific B cells identified in spleens from immunized mice (n = 5). (a) Identification of Pfs230D1B cells using a tetramer approach. Cells were gated in singlets, live, B220+, CD19+, GL7 + and after exclusion of unspecific binding by decoy antibody, cells were gated specifically in response to Pfs230D1 antigen, with the biotin-streptavidin tetramer expressing fluorochrome PE. Gating strategy is shown in Supplementary Fig. 1 (b) Frequency of Pfs230D1-specific B cells among 300,000 splenocytes.

Fig. 6

Standard Membrane Feeding assay with mouse sera to assess oocyst reduction after Pfs230D1 immunization and helminth infection. Sera from each experimental group was pooled (n = 10) and 100 µL was fed to mosquitoes along with cultured P. falciparum gametocytes. Counting of P. falciparum oocysts was performed in mosquito midguts and the TRA was calculated compared to the group fed gametocytes along with sera from naïve mice. One Way ANOVA and multiple comparison test revealed no difference in the TRA of post-vaccination sera from uninfected versus infected mice (p = 0.985), but significantly greater activity in both groups versus unvaccinated control mice. These results were generated with data from three independent SMFAs: two of them performed with pooled sera from 5 different mice for each experimental group, and the third experiment was performed with 10 pooled sera combined from the two previous experiments. ^*p < 0.05 compared to naïve group. Data are shown as mean + SEM.