B cell clonal expansion and mutation in the immunoglobulin heavy chain variable domain in response to Pfs230 and Pfs25 malaria vaccines

Abstract

Malaria transmission-blocking vaccines induce antibodies that target Plasmodium in the mosquito vector. We recently reported that Pfs230 vaccine achieves superior activity to Pfs25 in humans. Here, we describe clonal expansion in the variable region of immunoglobulin heavy chains (VH) of antigen-specific single B cells collected from humans immunized with Pfs230D1-EPA or Pfs25-EPA conjugate vaccines formulated in Alhydrogel®. Based on studies of CD27+ memory B cells following Pfs230 vaccination, clonal expansion and somatic hypermutation was seen in four of five subjects. Pfs25 did not induce sufficient CD27+ cells for sorting; based instead on CD19+ Pfs25-reactive B cells, clonal expansion was only seen in two of five subjects. Clonal expansions and mutations in Pfs230-specific single B cells combined with the enhanced activity of Pfs230 antibodies by complement, might justify the outstanding activity of Pfs230D1 as a TBV candidate.

Graphical Abstract

Recent advances in malaria vaccine development have expanded the array of Plasmodium falciparum antigens in clinical trials (Duffy and Patrick Gorres, 2020), such as sexual-stage proteins expressed by the parasite in the mosquito vector. By targeting sexual-stage proteins, vaccines seek to reduce the rate of parasite transmission in the community. Malaria transmission-blocking vaccines (TBVs) act primarily by eliciting antibodies that neutralize sexual stage parasites (Sagara et al., 2018; Coelho et al., 2021) but mechanisms that promote antibody diversification during TBV immunization are not yet clear. Among the leading TBV candidates, P. falciparum surface 230 kD protein Pfs230 and Pfs25 have shown promising activity in preclinical and clinical studies (Goodman et al., 2011; Talaat et al., 2016; Sagara et al., 2018; Singh et al., 2019; Tachibana et al., 2019). Our group previously demonstrated that when Pfs25 or the first domain of Pfs230 (Pfs230D1) are conjugated to the Exoprotein A (EPA) carrier and formulated with the Th2 adjuvant Alhydrogel®, both vaccine candidates induce functional antibodies in murine and non-human primate models (Healy et al., 2021). However, Pfs25-EPA has shown modest activity in humans (Talaat et al., 2016; Sagara et al., 2018), while Pfs230 induces superior activity in healthy volunteers (Healy et al., 2021). Furthermore, antibody responses to Pfs230 vaccine occur earlier than for Pfs25 in humans: Pfs25 antibody arises after the second dose, while Pfs230D1 antibody does so after the first dose in some recipients (Healy et al., 2021). The functionality of both vaccines is strongly dependent on antibody activity (Sagara et al., 2018; Healy et al., 2021) and complement enhances the activity of Pfs230 antibodies (Coelho et al., 2021), but a full understanding of the superior functional immunogenicity of Pfs230 over Pfs25 remains to be elucidated.

The success of a malaria vaccine that is mediated by antibody is determined by the capacity of immunoglobulins secreted by B cells to recognize and bind to an antigen, and then to neutralize the parasite. A diverse B cell receptor (BCR) repertoire is generated by the variable, diversity and joining regions (VDJ) recombination process that occurs in early stages of B cell maturation, and by somatic hypermutation and selection of B cells with higher affinity for a given antigen during immunization or infection. Thus, deep sequencing of BCRs can be used as a tool to measure changes in the BCR repertoire following immunization, and to determine how these changes are related to functional activity (Galson et al., 2015, 2016; Truck et al., 2015). Although antibodies are encoded by sequences of light and heavy chains, analyses of the heavy chain are considered sufficient to determine the signature in response to vaccination (Xu and Davis, 2000; Zhou and Kleinstein, 2019), since most of the sequence variation and antigen binding is mediated by the heavy chain.

Here, we sought to analyze heavy chain variable (VH) regions of Pfs230- or Pfs25-specific single B cells (henceforth called Pfs25 or Pfs230 B cells) from 10 Malian adults receiving either Pfs25-EPA/Alhydrogel® or Pfs230D1-EPA/Alhydrogel® vaccine (ClinicalTrials.gov NCT02334462). Subjects received four 40 μg doses of Pfs230D1 or four 47 μg doses of Pfs25 at days 0, 28, 168, 530 of the study. Peripheral blood mononuclear cells (PBMCs) were collected 14 days after the last dose, at day 554. The clinical study was approved by the ethics review boards from the Faculté de Médecine, de Pharmacie et d’OdontoStomatologie (FMPOS) Bamako, Mali and the US National Institute of Allergy and Infectious Diseases (NIH, Bethesda, Maryland, USA). The safety and immunogenicity results of the vaccinations in US malaria-naïve subjects are reported elsewhere (Healy et al., 2021). B cell staining, gating strategy and flow cytometry sorting were performed as previously described (Coelho et al., 2021).

The less potent immunogenicity of Pfs25-EPA/Alhydrogel® compared with Pfs230-EPA/Alhydrogel® was reflected by the low proportion of Pfs25 B cells among CD27+ memory B cells, resulting in an insufficient number for flow cytometry sorting. Instead, we sorted Pfs25 B cells from the CD19+CD20+ total B cell population, while we sorted the more abundant Pfs230 B cells from the CD19+CD20+CD27+ population. The average percentage of Pfs230 B cells was 1.34% (min= 0.03%; max= 2.70%) among CD27+ memory B cells, while the average percentage of Pfs25 B cells was 2.18% among the CD19+CD20+ total B cell population (min= 0.70%; max= 3.90%) (Supplementary Fig. S1). Nested PCR targeting the VDJ region and sequencing of single B cells were performed by iRepertoire as previously described (Coelho et al., 2021). We obtained 105 VH sequences from Pfs230 B cells and 101 VH sequences from Pfs25 B cells from 10 subjects in total (five subjects in each of the two vaccine groups) (Supplementary Table S1).

B cells that acquire mutations during germinal center reactions can improve antibody-antigen binding and are selected for further expansion, leading to affinity maturation of the antigen-specific B cell population (Kepler and Perelson, 1993). Thus, analyses of somatic hypermutation can reveal the expansion of clones that are related to higher antibody affinity. We performed analyses to evaluate clonal expansion in those BCR sequences using the R package Alakazam (Nouri and Kleinstein, 2020). Sequences were defined as being part of the same clonal expansion if they shared the same V and J genes, and were >90% similar in their CDR3 sequence. Data were submitted to Mendeley Data (doi: 10.17632/4xhwkd3jfx.1),

As expected by the sorting of CD27+ cells for Pfs230 and CD19+CD20+ for Pfs25, our results revealed that clonal expansions occurred with more frequency among Pfs230 B cells than for Pfs25 B cells, evidenced by a higher number of clusters (Fig. 1) and larger sizes of clusters that diverged from germline sequences (Fig. 2A). In addition, the number of mutations in the VH region was higher in Pfs230 B cells than Pfs25 B cells (Fig. 2B).

Fig. 1.

Lineages of Plasmodium falciparum surface 230 kD protein Pfs230 or Pfs25-specific clonal expansions. Sequences were defined as being part of the same clonal expansion if they shared the same V and J genes, and were >90% similar in their CDR3 sequence. Bold numbers represent subject ID and are shown above the cluster name. Black circles represent germline sequences, gray circles inferred sequences, and green circles the ID number of the heavy chain variable domain (VH) sequences identified. Numbers in the lines represent number of somatic hypermutations compared with the ancestor. Maximum parsimony tree was drawn using the R package alakazam.

Fig. 2.

Clonal expansion analyses of antigen-specific single B cells from subjects immunized with Plasmodium falciparum surface 230 kD protein Pfs230D1-ExoProtein A (EPA)/Alhydrogel Pfs25-EPA/Alhydrogel. (A) Cluster sizes and isotypes of the sequences. (B) Numbers of mutations in the heavy chain variable domain (VH) region. nt, nucleotide.

Pfs230 vaccine yielded a higher number of mutations in the IgM repertoire of CD19+CD20+CD27+ Pfs230 B cells compared with CD19+CD20+ Pfs25 B cells (Fig. 2, although this could be explained by the sorting of different populations. Three sequences from Pfs230 B cells were determined to be IgA.

Age groups differ in their IgM and IgA repertoire responses to pneumococcal vaccination (Wu et al., 2012), but here we used samples from subjects within a similar age range (Pfs230 = 25–40 years, average= 34; Pfs25= 25–51 years, average= 42). Thus, we believe the differing mutation profiles within antibody isotypes are the result of antigen-specific stimulation effects. IgA responses can be elicited in response to malaria vaccines, but as we determined here, they usually comprise a small proportion of the antibody repertoire (Biswas et al., 2014; Feng et al., 2018).

Three subjects from the Pfs25 group and one from the Pfs230 group showed no evidence of clonal expansion, although the only Pfs230 subject without evidence of clonal expansion had just two sequences analyzed (Supplementary Table S1). Previous data showed that immunization with infectious P. falciparum sporozoites under cover of chloroquine prophylaxis promoted strong B cell clonal expansion in five out of eight subjects (Murugan et al., 2018). All clusters determined in the Pfs230 B cells from subject 1 were shared with other subjects, totaling four convergent clusters (Supplementary Table S2), versus three convergent clusters for Pfs25 (Supplementary Table S3). We did not find a direct correlation between antibody titers or functional activity and clonal expansion, although subject 1 developed the highest number of clusters (Fig. 1) and had the highest antibody titers among Pfs230 vaccinees (Supplementary Table S4).

A caveat for interpretation of our study involves the comparison between Pfs25 B cells from the CD19+CD20+ population with Pfs230 B cells from the CD19+CD20+CD27+ population. We would expect higher mutation rates in CD27+ cells compared with CD19+20+ cells. However, this reflected the dearth of CD27+ cells induced in response to Pfs25. Further, we used antigen tetramers to sort antigen-specific B cells and confirmed the specificity of the probe for both antigens (Coelho et al., 2020, 2021).

In conclusion, Pfs230D1 induces a greater number of CD27+ cells than Pfs25, and a clonal expansion of antigen-specific B cells in four out of five subjects. This, allied to the enhanced activity of Pfs230 antibodies by complement, might contribute to the excellent activity of Pfs230D1 as a TBV candidate (Healy et al., 2021).

Highlights.

• Previously, we showed that immunogenicity and activity of Pfs230D1 vaccine is superior to Pfs25 in humans.

• Clonal expansion in IG heavy chains (VH) of Pfs230 B cells occurs with high frequency in response to Pfs230D1 vaccine.

• A stronger B cell response might contribute to superior Pfs230D1 activity.