One strategy towards the pre-erythrocytic stage is to target the parasite during the short span of time that the sporozoites are in the bloodstream. This sporozoite vaccine must induce the production of protective antibodies that will block and neutralize the sporozoites from invading liver cells. The other strategy is to target the sporozoites once they are inside the liver cells through the induction of CTLs that will destroy sporozoite infected liver cells.

Another approach is to induce blocking antibody towards the circulating merozoites, preventing them from infecting red blood cells. Once inside the erythrocytes, CTL cannot be generated against them since red blood cells do not express MHC molecules on its surface. However, some malaria antigens are expressed on the surface of the infected RBCs toward which antibodies can be directed against and be used for opsonization and complement. Also, it may be helpful to induce antibodies that block the infected erythrocytes from adhering to the lining of blood vessels. It is during the erythrocytic stage that illness associated with malaria occurs. There are strategies, called 'anti-disease' vaccines, towards the toxic products produced during this phase.

There are also attempts to produce a 'transmission-blocking' vaccine. This approach targets the sexual stage gametocytes of malaria. The goal is to prevent the gametocytes from producing more sporozoites within the gut of the mosquito vector, thus blocking the transmission of malaria. This vaccine does not prevent illness in an infected host, but it may be important to reduce the spread of malaria.

Creating a malaria vaccine does not only involve discovering the optimal antigens; it is also important to enhance the immune response towards those antigens through adjuvants, especially since adjuvants are usually required with non-living vaccines. Studies have shown that one of the main determinants of protection against malaria may be the adjuvant vehicle. The adjuvants may be essential in influencing the specificity and isotype of the desired antibodies. One study investigated the effectiveness of the CS protein conjugated to BSA in differing ratios. Even though antibody titers were comparable among different ratios, that did not correspond with induced protection. It seemed that certain peptide-carrier ratios elicited antibodies with greater avidity to the desired antigen.

Freund's complete adjuvant has been relatively successful as an adjuvant for malaria vaccines, but this adjuvant is too toxic for human use. Other options for adjuvants may be mycobacterial cell wall skeleton, monophosphoryl lipid A, squalene, and liposomes. There has been much recent clinical success with SBAS2.1/SBAS2, which are coupled with MSA-1 in vaccine form. These adjuvants have a greater potency than Freund's adjuvant.

Cytokine injection in conjunction with vaccine administration has proven limited success.

SPf66 - the first recognized malaria vaccine. SPf66 was developed in Colombia by Manual Patarroyo in 1987 by purifying three merozoite-derived proteins and joining them with sequences derived from the repeat domain of the CS protein of P. falciparum. Phase I trials demonstrated a 75% rate of efficacy and showed that the vaccine was immunogenic and well tolerated. Phase IIb and III trials demonstrated an efficacy rate that ranged between 38.8-60.2%. The first trial in Africa was conducted in Tanzania in 1993, where intense malaria transmission occurs. The estimated vaccine efficacy rate was 31% after a one-year follow-up period. SPf66 was also confirmed to be safe and immunogenic. A later trial in the Gambia did not show any protective effect elicited by the SPf66 vaccine; however, factors such as the short three and a half months follow-up period were given as reasons for this outcome.

In recent studies, it has been determined that SPf66 has very low immunogenicity and induces only a temporary humoral immune response (6 months on average). The antibody subtype implicated is IgG. Among African children, the antibody titer following exposure to SPff66 rises with age, however this finding is tentative.

There is much controversy surrounding the SPf66 vaccine. Many have criticized the manner in which the trials have been conducted and the fact that it is not understood how SPf66 mediates protection. SPf66 will most likely not be the answer to the malaria burden, but it has given several research teams valuable experience in conducting malaria vaccine trials that can be helpful during the next generation of trials.

CSP - a vaccine based on the circumsporozoite protein (CSP) has been developed and field tested in humans in a malaria-endemic region of Kenya (reference). The vaccine incorporated the recombinant (Asn-Ala-Asn- Pro15 Asn-Val-Asp-Pro)2-Leu-Arg (R32LR) covalently linked to purified Pseudomonas aeruginosa toxin A 9. The intended outcome was an elevated T-lymphocyte response, however this was not observed in the study group. There was no observed reduction in incidence of disease in the study group as compared to the control group (which received hepatitis B vaccine). CSP vaccine recipients had an 82% incidence of parasitemia, while the control group had an 89% incidence. Therefore, this study concluded that CSP vaccine-induced anti-sporozoite antibody was not protective.

In rat models, subunit CSP-based vaccines are being tested. It is understood that a vaccine which incorporates antigen drawn from numerous sub-species of falciparum will confer the maximum protectivity and ensure that vaccines are not limited by geographical constraints. Furthermore, DNA vaccines against malaria have incorporated CSP sequencing genes.

NYVAC-Pf7 - A recently developed multistage vaccine, NYVAC-Pf7 is a single NYVAC genome containing genes encoding seven Plasmodium falciparum antigens. Of these antigens, two are derived from the sporozoite stage of the parasite life cycle (CSP and sporozoite surface protein 2 (PfSSP2)), one from the liver stage (liver stage antigen 1 (LSA1)), three from the blood stage (merozoite surface protein 1 (MSP1), serine repeat antigen (SERA), and AMA-1), and one from the sexual stage (25-kDa sexual- stage antigen (Pfs25)). The intent is that the use of multiple antigens from P. falciparum will induce immunity in recipient.

Trials with rhesus monkeys were carried out with promising results: specific antibody responses against four of the seven antigens were observed. These were the CSP, PfSSP2, MSP1, and Pfs25. The purpose behind the use of a poxvirus-based vaccine was to maximize the elicitation of cellular immunity.

In phase I/IIa safety, immunogenicity, and efficacy vaccine trials in humans (1998), vaccine was tolerated by variably immunogenic. Antibody responses were generally poor, however, cellular immune responses were detected in well over 90% of the test subjects. In some cases, administration of the vaccine conferred complete protection to falciparum challenge. Further trials are under way.

[NANP]19-5.1 - A field trial with the recombinant P. falciparum vaccine [NANP]19-5.1 yielded promising results in 1995. [NANP]19-5.1 consists of 19 repeats of the sporozoite surface protein [NANP] and the schizonts export antigen 5.1. Of the 194 school children vaccinated in this study, no child developed clinical malaria within a 12 week observation period and that all but 8 had considerably higher levels of antibody to both antigens.

However, this vaccine is limited by the fact that it contains no immunodominant T-cell epitopes. In its current form, the vaccine is only 20% peptide and has limited immunogenicity. The use of recombinant IL-2 adjuvant in conjunction with this vaccine has proven to be successful.

RTS, S - is a recombinant vaccine consisting of the circumsporozoite protein found on the surface of the sporozoite stage of Plasmodium falciparum. This antigen elicits antibodies that are capable of preventing sporozoites from invading hepatocytes, and a cellular response that is capable of eliminating infected hepatocytes. The problem with the circumsporozoite protein is that it is poorly imunogenic. Therefore, in the RTS, S vaccine, the circumsporozoite protein is fused with a hepatitis B surface antigen, which creates a much more potent vaccine. This vaccine was combined with either alum and monophosphoryl A or only an oil in water emulsion only prevented infection in 1 in 8 and 2 in 7 respectively. However, an emulsion of oil in water, and the adjuvants monophosphoryl A and QS21 (SBAS2) prevented infection in 7 out of 8 volunteers challenged with P.falciparum.

In 1998, a human subject study concluded that in addition to superior humoral responses, vaccinated individuals demonstrate extreme T-cell proliferation and IFN-gamma production. The current limitation is that conferred immunity rapidly declines following a six-month period. The improvement of vaccine composition, use of adjuvants, and an improved immunization schedule offer hope to confer longer-lasting protective immunity.

Pfs230 - This sexual-stage falciparum surface antigen can elicit antibodies which block the infectivity of gametes to mosquitoes. The 360-kDa protein is localized to a the parasitophorous vacuole/parasite plasma membrane. In a 1997 study, it was shown that sera which mediate gamete lysis contain IgG1 and IgG3 antibodies to gamete surface proteins. Thus, Pfs230 is a major target of C'-fixing antibodies.

It appears, then, that multi-component DNA vaccines offer the best prospects for protection against malaria and subsequent cerebral malaria development. They can be tailored to include a variety of numbers and types of epitopes, and the arrangement of these epitopes can be altered as well. In addition, there are numerous other advantages of DNA vaccines over conventional vaccines, including high immunogenicity, modifiability, stability, and cost. Recent large-scale Phase I/II clinical trials of Spf66 and NYVAPf-7, two multi-component synthetic peptide malaria vaccines, revealed only limited protection. Thus, the need for DNA-based vaccine technology is apparent. The efficacy of these future DNA vaccines may be increased by adjuvant use. Shi et al demonstrated that adjuvants can have a significant influence on antibody response activity when used in conjunction with vaccines. Freund’s adjuvant was shown to produce higher level of antibodies, while block-copolymer adjuvant induced higher affinity antibodies.