Both vaccines investigated in this study were well tolerated, although local adverse events were more frequent and severe when CPG 7909 was added and more severe when higher doses were given. Neutropenia and malaise was more frequent in the CPG groups. A previous study, in which a blood stage malaria vaccine (AMA1-C1/Alhydrogel) was given to malaria naïve adults with and without CPG 7909, showed an increase in local adverse events and greater severity of local and systemic events with CPG, and several volunteers were withdrawn due to adverse events 
. However, a subsequent Phase 1 study in malaria naïve adults showed a more favorable adverse event profile, and in Malian adults minimal local reactogenicity and no related systemic reactogenicity was seen 
. In this study only one Grade 3 adverse event related to vaccination occurred (severe erythema), and no volunteers were withdrawn due to adverse events. Transient neutropenia occurred in the groups receiving CPG, typically 3 days after vaccination with a return to baseline levels by Day 7. Transient neutropenia has been observed in previous studies of CPG 7909 administration and is believed to be due to immune stimulation and white blood cell sequestration 
. As in previous studies, no clinical events occurred related to this neutropenia and its relevance as a safety signal in this population is doubtful.
Autoimmune disease is a theoretical concern with administration of CPG adjuvants. This concern has been heightened since the occurrence of Wegeners Granulomatosis (an autoimmune vasculitis affecting the lungs and kidneys) in a subject who received a recombinant hepatitis B vaccine adjuvanted with ISS, an oligonucleotide similar to CPG 7909 
. However, the causality of a single occurrence in an early phase clinical trial is difficult to assess, and vasculitis has also been previously reported with licensed hepatitis B vaccines 
. In this study no volunteers developed laboratory markers of autoimmune disease, despite the pre-existence of low-positive ANA in some volunteers, and no clinical autoimmune events occurred. To date, including this study, a total of 111 volunteers have received malaria antigens adjuvanted with Alhydrogel and CPG 7909 
. While this number is far too small to evaluate the risk of autoimmune disease or other rare adverse events, the marked enhancement of antibody responses with CPG must also be considered when weighing potential risk and benefit, particularly for a vaccine intended to prevent morbidity and mortality due to P. falciparum
malaria. The safety profile in the target population, malaria exposed children, is as yet unknown.
Antibody is thought to be the primary mechanism of protection against blood stages of malaria 
. A previous study of AMA1-C1/Alhydrogel with and without CPG 7909 showed up to 14-fold enhancement of antibody responses when CPG was added 
. In this study the addition of CPG also markedly enhanced antibody responses, with a 49.4 fold enhancement seen after second vaccination with the 40 µg dose compared to the group not receiving CPG. Also as in the previous study with AMA1-C1/Alhydrogel, responses in the groups receiving CPG peaked after the second vaccination, with no additional increase after the third vaccination, and responses were similar in the high and low dose groups when CPG was added. Thus CPG causes a dose sparing effect, where responses after two doses with 40 µg antigen are higher than responses after three doses with 160 µg without CPG, and are similar to two doses with 160 µg antigen with CPG. This dose sparing effect is particularly important in a strategy where multiple antigens are combined, as may be required for an effective blood stage malaria vaccine. Faster induction of antibody responses is also an advantage, and has been seen when CPG is added to hepatitis B vaccine 
. Significant enhancement of antigen specific memory B cell responses was seen after vaccination with malaria antigens with CPG, consistent with the marked enhancement in antibody responses 
While no assays have been validated as surrogates of protection for malaria vaccines, in vitro GIA has been utilized to measure the biologic activity of antibodies elicited in clinical trials of blood stage antigens 
. In vitro growth inhibition of up to 96% has been demonstrated with AMA1 vaccines 
. Despite the high levels of antibody induced here, comparable to those induced when CPG was added to AMA1-C1/Alhydrogel 
, the maximum level of growth inhibition induced in any volunteer was only 32%. A previous study has shown that more anti-MSP142
antibody is required to reach the same level of inhibition in GIA compared to anti-AMA1 antibody 
, thus although higher antibody (and correspondingly higher GIA) was induced with the addition of CPG, a level of inhibition likely to be biologically significant was not achieved in any volunteer. A recent study suggested that Fc receptor-mediated protection is important in MSP1-induced immunity 
. This protective mechanism cannot be measured by GIA. Therefore, in the case of MSP142
-based vaccines investigators may need to consider other assay(s) to evaluate the biologic function of the induced antibody.
A Phase 2b study in Kenyan children of a monovalent MSP142
vaccine adjuvanted with AS02 failed to show protection against clinical malaria 
. Geometric mean antibody levels induced by vaccination in that population were ~23 µg/mL, less than those induced in malaria naïve adults in this study. Strain-specific effects have been demonstrated in an Aotus
model when animals received an MSP142
vaccine with complete Freund's adjuvant 
, and may also have played a part in the lack of benefit demonstrated in Kenyan children. The same Aotus
study showed no protection with an MSP142
vaccine adjuvanted with AS02, but showed some protection with the same vaccine in a water-in-oil adjuvant (ISA 720), and the authors speculate that a Th1-biased adjuvant such as CPG could be added to induce additional protection.
It seems likely that a recombinant blood stage malaria vaccine will require multiple proteins, both to overcome diversity of the allelic antigens expressed by the parasites and to overcome diversity of responses in the target population. Which combination of antigens is most likely to be protective is as yet unknown, with the epidemiologic evidence conflicting 
. Clinical development of blood stage vaccines is largely empiric, since correlates of protection are unknown. Vaccines which have been judged to be safe and adequately immunogenic have gone forward to Phase 2b trials in African children, but given the lack of protection demonstrated to date this approach may no longer be feasible. A human blood stage challenge using parasite-infected erythrocytes is under development and may prove useful in allowing down selection of blood stage vaccine candidates prior to large field trials 
. While further clinical development of MSP142
/Alhydrogel + CPG 7909 as a separate candidate vaccine is not currently anticipated, the good safety profile, high antibody responses, and biologic function of the induced antibody demonstrated in this trial provide a rationale for the inclusion of MSP142
/Alhydrogel + CPG 7909 as part of a combination blood stage vaccine, which is currently in early phase clinical development (ClinicalTrials.gov Identifier: NCT00889616). Demonstration of homologous protection in either a human challenge or non-human primate model (using adjuvants suitable for use in humans) would provide support for continued clinical development, although such models are as yet unvalidated with respect to their ability to predict protection in the target population. Demonstration of likely benefit may be of particular importance when novel adjuvants with a limited safety profile, such as CPG 7909, are part of the clinical formulation.