Abstract:
Despite being treatable and preventable, malaria kills about half a million people each year,
mostly in Africa. Notwithstanding many attempts to develop a malaria vaccine, only the
vaccine RTS,S/AS01 (Mosquirix), has been fully developed and was recommended by the
World Health Organization for use in children in malaria endemic areas on October 6th in
2021. RTS,S is a subunit vaccine and provides only partial protection. Manufacturing of
pharmaceutical grade Plasmodium falciparum (Pf) sporozoites (SPZ) has been achieved in the
past decade, making the development of plasmodial whole cell vaccines possible. This may be
a novel and better platform to design malaria vaccines. In addition, the availability of
cryopreserved infectious sporozoites has boosted possibilities to assess vaccine efficacy by
using controlled human malaria infection (CHMI) trials. The most effective vaccination
strategy to date is to inoculate PfSPZ while using chemoprophylactic antimalarials such as
chloroquine – the PfSPZ Chemoprophylaxis Vaccine (PfSPZ-CVac). Although CHMI can test
vaccine efficacy (VE), the immune mechanisms underneath are not fully understood.
Particularly, the cellular immune response is poorly characterised compared to the humoral
response (i.e. antibody titers). Thus, the systematic monitoring of cellular immune responses
during immunization in combination with CHMI might be a valuable step to guide the clinical
development of malaria vaccine candidates and identify immune mechanisms causally related
to protection.
In Chapter 1, I investigated the influence of two accelerated regimens of PfSPZ-CVac on the
generation of pro-inflammatory Pf-specific CD4+ T helper cells and their suitability as a
surrogate of protection. The trial was conducted in healthy, malaria-naïve adults in Tübingen.
To detect antigen-specific cells in peripheral blood with increased sensitivity, Plasmodium specific T cells were enriched by magnetic-activated cell sorting followed by staining and
multiparameter flow cytometry (MFC). Measurements were done using the flow cytometer
FACS Canto II and stimulations were done with infected red blood cells (iRBCs). Uninfected
red blood cells and Staphylococcal enterotoxin B (SEB) were also as negative and positive
controls, respectively. Unexpectedly, I found that a condensed vaccination schedule, where the
three vaccinations were given within 10 days induced higher frequencies of Plasmodium specific CD40L+CD4+ TNF-α+/IFN-γ+ cells than a 28-day regimen. The response was also
qualitatively different. The shorter regimen led to a polarized Th1 response with more CD45RO+CCR7- effector memory T cells. This may lead to higher numbers of memory cells
in the liver.
A tendency towards higher frequencies of specific CD40L+CD4+ TNF-α+/IFN-γ+ effector
memory T cells was present in protected volunteers but no reliable correlate of protection could
be identified. Future research will be needed to identify effector and regulatory responses that
predict vaccine efficacy. It will be particularly important to include the characterization of
Plasmodium-specific cytotoxic T cells.
More than 94% of the estimated malaria cases globally occur in Africa. Therefore, every
malaria vaccine must be tested in malaria exposed volunteers to be sure that it has a significant
public health impact. Unfortunately, for many vaccines, VE is lower in Africa. This effect is
particularly strong in malaria vaccines. The GMZ2 vaccine was developed to prevent malaria
and its complications by mimicking naturally acquired immunity. The vaccine antigen consists
of a fusion protein between fragments of the merozoite surface protein-3 and the glutamate rich protein. In Chapter 2, I investigated the immunogenicity of GMZ2 adjuvanted with two
different immune modulators: Alhydrogel or CAF01. The study was performed in healthy,
adult, lifelong malaria-exposed volunteers from Lambaréné, Gabon. MFC was used to
systematically measure the T and B cell response, and to compare immune response patterns
before and after immunization. Peripheral blood mononuclear cells were cryopreserved and
measured upon completion of the trial using a Sony SP6800 Spectral Analyzer. Monitoring of
GMZ2-stimulated CD4+ T, and CD20+ B cells showed that GMZ2 did not induce significant
immune responses beyond the baseline. The low response to vaccination was unexpected as
was the similar performance of the two adjuvants. VE in CHMI was similar between the
groups, hence these findings may be expected. Notwithstanding the negative result, this study
will help guiding the development of the next generation of blood stage malaria vaccines in
malaria-exposed volunteers, where baseline responses play a major role in generating
successful immune responses following vaccination.
In summary, the work presented as part of the thesis shows that systematic monitoring of the
cellular immune responses by MFC, in combination with CHMI studies is a valid, and stringent
approach to measure VE and identify correlates of protection, surrogate markers, and the effect
of schedule and pre-existing immunity on vaccine responses. More work will be required to
replace CHMI with immunological surrogate endpoints and understand vaccine-induced
antimalarial immunity.
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