RTS,S/AS01E is the only malaria vaccine recommended for widespread use by the World Health Organization (WHO) in African children1. The vaccine has a favorable safety profile and reduces episodes of both clinical and severe malaria in children2. Data from the phase 3 trial showed that vaccine efficacy against clinical malaria is modest 12 months after a 3-dose primary vaccination, estimated at 55.8% (97.5% confidence interval [CI] 50.6–60.4) in children age 5–17 months2, and 31.3% (23.6–38.3) in infants age 6–12 weeks3, and the duration of protection wanes over time2,3. The development of a more efficacious vaccine remains a high priority for malaria control and elimination. Multiple factors may contribute to the suboptimal efficacy of adjuvanted recombinant protein subunit vaccines like RTS,S/AS01E, and the absence of native glycosylation residues may be one important element4. The vaccine, produced by GlaxoSmithKline Biologicals (Belgium), contains the hepatitis B virus surface antigen (HBsAg) genetically fused to a fragment of the Plasmodium falciparum circumsporozoite protein (PfCSP) including the last 18 NANP repeats of the central domain and its C-terminal end, and is formulated as virus-like particles with a liposome-based adjuvant (AS01E)5. We have previously shown that the avidity of antibodies to the C-terminus of CSP correlates with protection against malaria following vaccination with RTS,S/AS01E6.
The CSP C-terminus comprises a domain with homology to the thrombospondin type-1 repeat superfamily (TSR), which in its native form contains an O-fucosylation motif7, and a fucose monosaccharide modification was recently identified on PfCSP from salivary gland sporozoites8. O-fucosylation is a simple post-translational modification that facilitates protein folding and trafficking and may affect antigenicity7. The role of O-fucosylation in the P. falciparum life cycle and the interactions with its divergent hosts have only started to emerge. Recent evidence suggests that O-fucosylation of TSR is important for P. falciparum liver infection and may also be relevant in the mosquito stages of the life cycle9. Moreover, the glycosylation profile on P. falciparum surface proteins such as PfCSP has the potential to influence parasite-specific humoral and cellular immune responses, particularly if the glycosylated antigen represents an important T cell epitope4. Immunization elicits CD4+ T cell immune response to PfCSP10, aiding in antibody production by B cells. We, therefore, hypothesized that the antibodies induced by the RTS,S vaccine, in which PfCSP is not fucosylated, might not properly recognize the native fucosylated CSP antigen on the sporozoite, which could compromise the overall protective efficacy. To test this hypothesis, we used immune sera from young children enrolled in the pivotal Mozambique phase 2b trial of the RTS,S vaccine formulated with a previous version of the adjuvant (AS02A)11, and compared the levels of vaccine-induced IgG that bound to fucosylated vs. non-fucosylated CSP.
The baseline characteristics between vaccination groups (Table S1) and pre-vaccination antibody levels (Month[M] 0) were similar for both fucosylated and non-fucosylated versions of PfCSP full-length (Figure 1A) and C-terminus TSR domain-only antigens (Figure 1B). Primary vaccination (M3) anti-PfCSP IgG reactivity was strong, with fold-increases in geometric mean levels >1000 for RTS,S vaccinees as compared to the comparator group. These data are well-aligned with previous estimates using ELISA for a larger niched study of the same clinical trial12 and with other RTS,S clinical trials measuring anti-NANP IgG13. Anti-TSR IgG responses were somewhat weaker, with 5-6-fold reductions between vaccination groups at M3, compared to full-length PfCSP. Also, as expected, PfCSP full-length and TSR antibody levels waned between M3 and M8 (reductions by 5 and 3.5-fold, respectively), closely replicating previous estimates of IgG decay rates12. Contrary to our expectation, we observed nearly identical responses when we compared IgG levels bound to fucosylated PfCSP full-length and TSR antigens vs. non-fucosylated constructs. Figure 1 (C and D) shows anti-PfCSP fucosylated vs. not fucosylated IgG levels, yielding a bivariate distribution that approached perfect correlation. Next, we examined the factors affecting RTS,S/AS02A antibody level increases relative to pre-vaccination. We observed no significant differences in the rise of IgG level geometric means between pre- and post-vaccination time points against the fucosylated PfCSP proteins across age groups or sexes in the RTS,S vaccinated children, except for a lower increase of TSR fucosylated IgG levels in older (2-4 years) compared to younger (1-2 years) children from M0 to M3 (p=0.02, Table 1). As expected, higher increases in anti-PfCSP full-length and TSR IgG levels upon vaccination were associated with protection from disease. Thus, RTS,S-vaccinated children who did not develop clinical malaria within the first year of follow-up had significantly higher anti-PfCSP IgG increases from M0 to M3 (p=0.042, Table 1) as well as a trend towards higher anti-TSR IgG increases (p=0.2). Differences were larger and more clearly significant for anti-PfCSP IgG level increases from M0 to M8 for both full-length (p=0.0004) and TSR (p=0.005) (Table 1). Associations between IgG levels against non-fucosylated PfCSP full-length and TSR antigens with age, sex, and clinical malaria, were identical to the results reported for fucosylated antigens (data not shown). To determine whether malaria endemicity could influence the binding of vaccine or naturally induced IgG to fucosylated vs. non-fucosylated PfCSP antigens, another set of samples (n=124) from Ilha Josina, a trial site of higher transmission intensity (cohort 2)11 was also tested. Again, no difference was noted in recognition of fucosylated compared to non-fucosylated PfCSP antigens (data not shown), suggesting that the binding is independent of malaria exposure.
To our knowledge, no previous study has directly tested whether RTS,S vaccine-induced IgG antibodies bind less to a “native-like” fucosylated PfCSP (present on P. falciparum sporozoites)8 than to non-fucosylated PfCSP (as present in the RTS,S vaccine). Of note, while our results show that RTS,S-induced antibodies recognize similarly fucosylated and non-fucosylated PfCSP constructs, we observed the same phenomenon in pre-vaccination and comparator post-vaccination samples. These data suggest that IgG induced by natural exposure, presumably to fucosylated PfCSP on sporozoites, efficiently recognized the target antigen epitopes regardless of fucosylation. Nevertheless, we cannot rule out a potential benefit of an alternative RTS,S vaccine containing fucosylated PfCSP. To establish this, fucosylated and non-fucosylated PfCSP-based vaccines should be tested head to head to compare overall immunogenicity (humoral and cellular responses) and dose-dependent functional antibody titers that could mediate vaccine efficacy. The quality of immunoglobulins might differ when induced with a fucosylated PfCSP, particularly IgG subclasses, their avidity, and their neutralizing and non-neutralizing functional capacities. It is also possible that a fucosylated PfCSP protein-based vaccine could lead to broader glycan-dependent T helper and cytotoxic cell responses, as it can be presented on both MHC-I and MHC-II molecules, and the resulting complex can be recognized by T cells14. Recent advancements in human immunodeficiency virus (HIV-1)15 and Hepatitis C virus (HCV)16 vaccine development have been stimulated by a better understanding of the HIV-1 envelope spike and HCV-(E1 and E2) glycan composition and their interactions with the human immune responses. These advances highlight increasing evidence that glycans are important antigenic determinants in immune responses to various pathogens. Their relevance for prophylactic and therapeutic vaccine design and development is worth exploiting in more depth.
In conclusion, our data suggest that post-translational modification by O-fucosylation in PfCSP, absent in the RTS,S vaccine, does not affect antibody-antigen binding. However, this study does not rule out the possibility that a fucosylated PfCSP-based vaccine could generate functionally different antibodies that could be more effective anti-parasitic effectors, thus affecting the overall protective immunity.
Table 1. Factors affecting increases in IgG antibody levels reacting to fucosylated PfCSP constructs. Associations are reported as ratio estimates of month (M)3/M0 or M8/M0 between groups of interest (compared conditions).
|
|
M3/M0
|
M8/M0
|
Antigen
|
Compared conditions
|
Fold Difference
|
p-value
|
Fold Difference
|
p-value
|
PfCSP full-length
|
Increase Ratio Estimate
|
5079.1 CI [3833.1, 6730.01]
|
<0.0001***
|
829.2 CI [631.9, 1088.2]
|
<0.0001***
|
Old/Young
|
0.65 CI [0.34, 1.25]
|
0.20
|
0.61 CI [0.32, 1.14]
|
0.12
|
Female/Male
|
1.04 CI [0.59, 1.83]
|
0.89
|
0.83 CI [0.48, 1.43]
|
0.48
|
Malaria not infected/infected
|
1.96 CI [1.02, 3.75]
|
0.042*
|
3.07 CI [1.64, 5.73]
|
0.0004**
|
PfCSP TSR
|
Increase Ratio Estimate
|
778.4 CI [625.7, 968.3]
|
<0.0001***
|
153.3 CI [128.0, 183.5]
|
< 0.0001***
|
Old/Young
|
0.56 CI [0.34, 0.92]
|
0.02*
|
0.7 CI [0.46, 1.06]
|
0.09
|
Female/Male
|
1.1 CI [0.71, 1.7]
|
0.68
|
0.85 CI [0.59, 1.21]
|
0.36
|
Malaria not infected/infected
|
1.39 CI [0.84, 2.31]
|
0.2
|
1.8 CI [1.18, 2.72]
|
0.005*
|
Abbreviations: M0, month 0; M3, month 3; M8, month 8; CSP, Circumsporozoite protein; TSR,Thrombospondin type-1 repeat.
*p<0.05; **p<0.01, ***p<0.001.