Highlights d We isolate a non-neutralizing antibody from PyCSPimmunized BALB/cJ mice d A non-neutralizing Ab, RAM1, abrogates the blocking capacity of an aCSP NAb d A non-neutralizing Ab, RAM1, reduces the vaccine efficacy in mice in vivo d Further exploration for the role of pre-existing nNAbs in the field is warranted

Blocking Plasmodium, the causative agent of malaria, at the asymptomatic pre-erythrocytic stage would abrogate disease pathology and prevent transmission. However, the lack of well-defined features within vaccine-elicited antibody responses that correlate with protection represents a major roadblock to improving on current generation vaccines. We vaccinated mice (BALB/cJ and C57BL/6J) with Py circumsporozoite protein (CSP), the major surface antigen on the sporozoite, and evaluated vaccine-elicited humoral immunity and identified immunological factors associated with protection after mosquito bite challenge. Vaccination achieved 60% sterile protection and otherwise delayed blood stage patency in BALB/cJ mice. In contrast, all C57BL/6J mice were infected similar to controls. Protection was mediated by antibodies and could be passively transferred from immunized BALB/cJ mice into naïve C57BL/6J. Dissection of the underlying immunological features of protection revealed early deficits in antibody titers and polyclonal avidity in C57BL/6J mice. Additionally, PyCSP-vaccination in BALB/cJ induced a significantly higher proportion of antigen-specific B-cells and class-switched memory B-cell (MBCs) populations than in C57BL/6J mice. Strikingly, C57BL/6J mice also had markedly fewer CSP-specific germinal center experienced B cells and class-switched MBCs compared to BALB/cJ mice. Analysis of the IgG γ chain repertoires by next generation sequencing in PyCSP-specific memory B-cell repertoires also revealed higher somatic hypermutation rates in BALB/cJ mice than in C57BL/6J mice. These findings indicate that the development of protective antibody responses in BALB/cJ mice in response to vaccination with PyCSP was associated with increased germinal center activity and somatic mutation compared to C57BL/6J mice, highlighting the key role B cell maturation may have in the development of vaccine-elicited protective antibodies against CSP.

Both subunit and attenuated whole sporozoite vaccination strategies against Plasmodium infection have shown promising initial results in malaria-naïve westerners but exhibited less efficacy in malaria-exposed individuals in endemic areas. It has been hypothesized that preexisting immunity to malaria represents a significant roadblock to the development of a protective vaccine. Here, we demonstrate proof-of-concept that non-neutralizing antibodies (nNAb) can directly interfere with protective anti-PyCSP humoral responses. We developed and characterized a novel monoclonal antibody, RAM1, against the P. yoelii sporozoite major surface antigen, circumsporozoite protein (CSP). Unlike the canonical PyCSP repeat domain binding and neutralizing antibody (NAb) 2F6, RAM1 does not inhibit sporozoite traversal or entry of hepatocytes in vitro. Though 2F6 and RAM1 bind non-overlapping regions of the CSP-repeat domain, pretreatment with RAM1 abrogated 2F6’s capacity to block sporozoite traversal and invasion in vitro. Importantly, RAM1 reduced the efficacy of the polyclonal humoral response against CSP in vivo, paralleling the observed reduced efficacy of RTS,S in malaria-exposed populations. Taken together, our data demonstrate the interference of non-neutralizing antibodies with the efficacy of NAbs and may impact the efficacy of anti-CSP vaccines in malaria-exposed individuals.

Blocking Plasmodium, the causative agent of malaria, at the asymptomatic pre-erythrocytic stage would abrogate disease pathology and prevent transmission. Rodent-infectious species of Plasmodium such as P. yoelii (Py) serve as key tools to study vaccine efficacy and disease biology in immune-competent experimental animals. Here we evaluated the differences in vaccine-elicited humoral immunity in two widely used, and vastly diverged, inbred mouse strains, BALB/cJ and C57BL/6J, and identified immunological factors associated with protection. We vaccinated with Py circumsporozoite protein (CSP), the major surface antigen on the sporozoite, and evaluated protective efficacy after mosquito bite challenge. Vaccination achieved 60% sterile protection and otherwise delayed blood stage patency in BALB/cJ mice, whereas; all C57BL/6J mice were infected similar to controls. Interestingly, protection was mediated by antibodies, and could be passively transferred from immunized BALB/cJ mice into naive C57BL/6J. Dissection of the underlying immunological features of protection revealed early deficits in antibody titers and polyclonal avidity in C57BL/6J mice. Additionally, PyCSP-vaccination in BALB/cJ induced a significantly higher proportion of antigen-specific B-cells and class-switched memory B-cell (MBCs) populations than in C57BL/6J mice. Strikingly, C57BL/6J mice also had markedly fewer germinal center experienced, CSP-specific class-switched MBCs compared to BALB/cJ mice. Analysis of the IgG γ chain repertoires by next generation sequencing in PyCSP-specific memory B-cell repertoires also revealed higher somatic hypermutation rates in BALB/cJ mice than in C57BL/6J mice. These findings indicate that BALB/cJ mice achieved higher levels of B cell maturation in response to vaccination with PyCSP, which likely enabled the development of protective antibody responses. Overall, our study indicates that germinal center activity and B cell maturation are key processes in the development of vaccine-elicited protective antibodies against CSP.