Military personnel are an important target population for hepatitis A immunization. Soldiers are often given vaccines by jet injector and may be required to receive multiple vaccines at one time. Formalin-inactivated hepatitis A vaccine containing 360 ELISA units of antigen was evaluated at Fort Campbell. Volunteers received vaccine at 0, 1, and 6 months as follows: group 1, hepatitis A vaccine by needle; group 2, hepatitis A vaccine by jet injector; group 3, hepatitis B vaccine by needle; and group 4, both hepatitis vaccines by needle in separate arms. Immune response and reactogenicity were evaluated. After two doses, recipients of vaccine administered by jet injector had a higher prevalence of antibody than those who received vaccine by needle (93% vs. 79%). By the 8th month, the vaccine was 100% immunogenic by either route or with hepatitis B vaccine. No interaction between hepatitis A and B vaccines was detected.

Volunteers immunized with gamma-irradiated Plasmodium falciparum sporozoites serve as the gold standard for protective immunity against mosquito-borne malaria transmission and provide a relevant model for studying protective immune effector mechanisms. During a 7-12 month period, we immunized four volunteers via the bites of irradiated, infected mosquitoes. Following these exposures to attenuated sporozoites, all four volunteers developed antibodies to sporozoites as measured by an immunofluorescence assay and by an enzyme-linked immunosorbent assay using the circumsporozoite (CS) protein repeat-based molecule R32LR as capture antigen. Three volunteers also developed antibodies against the nonrepeating (flanking) regions of the CS protein; the level of these antibodies paralleled the serum activity to inhibit sporozoite invasion of hepatoma cells in vitro. These three volunteers were protected against malaria transmitted by the bites of five infected mosquitoes. Two of these protected volunteers received additional immunizing doses of irradiated sporozoites and were subsequently protected against challenge with a heterologous P. falciparum clone. No detectable fluctuations were observed in circulating levels of tumor necrosis factor, interferon-gamma, or interleukin-6 during the course of this study. Analysis of the humoral and cellular immune responses of these protected volunteers is expected to yield important clues to additional targets of immunity against the pre-erythrocytic stages of malaria parasites.

This study describes the safety and immunogenicity of a liposome-based vaccine injected into human subjects. Thirty healthy adult male volunteers were immunized with a liposome-encapsulated recombinant protein (R32NS181) containing epitopes from the repeat region of the circumsporozoite protein of Plasmodium falciparum. This antigen had previously been found to be poorly immunogenic in humans when it was adsorbed with Al(OH)3. In the present study, R32NS181 was encapsulated in liposomes containing monophosphoryl lipid A that were subsequently adsorbed to Al(OH)3. Increasing doses of liposomes containing antigen and monophosphoryl lipid A were used, but the liposomes were always adsorbed to the same dose of Al(OH)3. R32-specific serum IgG antibody responses to liposome-encapsulated R32NS181 were much higher than levels attained previously in humans with R32NS181 adsorbed to Al(OH)3. Geometric mean specific IgG levels after three doses ranged from 14 to 33 micrograms/ml. Sera from volunteers receiving the two highest doses inhibited P. falciparum sporozoite invasion of cultured hepatoma cells by an average of 92%, a result that was again superior to previously reported vaccines. Moderate but acceptable transient local reactogenicity was noted at high doses of the vaccine formulation, but little or no systemic toxicity was seen despite liposomal monophosphoryl lipid A doses up to 2200 micrograms. We conclude that encapsulation of poorly immunogenic circumsporozoite protein repeat peptides in monophosphoryl lipid A-containing liposomes is a successful adjuvant strategy in humans for inducing high levels of specific antibody production.

Control of hepatitis A has been an important concern for US military forces in war and peace. Immune serum globulin, although effective, is exceedingly cumbersome to use. The prevalence of antibody against hepatitis A is decreasing in young American soldiers, putting them at risk of hepatitis A during deployment. The US Army has been an active participant in development of hepatitis A vaccine. The first successful cell-culture-derived, formalin-inactivated hepatitis A vaccine was developed at the Walter Reed Army Institute of Research. This prototype vaccine was shown, in 1986, to be safe and immunogenic for humans. Since then we have evaluated the following issues related to the use of inactivated hepatitis A vaccines in military populations. Immunogenicity of vaccine derived from the CLF and HM175 strains; immunogenicity of hepatitis A vaccine given by jet injector; immunogenicity of hepatitis A vaccine when given with hepatitis B vaccine; immunogenicity when given in shortened schedules; safety and immunogenicity in Thai children; and efficacy under field conditions in the tropics. The hepatitis A vaccines which we tested are safe and highly immunogenic. Immunization by jet gun confers immunity equivalent to immunization by needle. Hepatitis A vaccine is equally potent when given with hepatitis B vaccine. Data on rapid immunization schedules and efficacy are under evaluation. We conclude that hepatitis A vaccine is a major improvement in our ability to prevent hepatitis A in soldiers.

Cytotoxic T lymphocytes (CTL) against the circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodium falciparum CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falciparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P. falciparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368-390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8+ T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B10.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria.

Human immune responses to modern synthetic and recombinant peptide vaccines administered with the standard adjuvant, aluminum hydroxide, tend to be poor, hence the search for better adjuvants. Antibody responses to a Plasmodium falciparum circumsporozoite (CS) protein vaccine, R32NS1(81), administered with an adjuvant containing cell-wall skeleton of mycobacteria and monophosphoryl lipid A in squalane (MPL/CWS) have been compared to responses to the same immunogen administered with aluminum hydroxide. 2 weeks after the third dose the following indices were greater in the 5 patients who received MPL/CWS than in controls (p less than 0.05): the geometric mean concentration (2.0 vs 25.4 microgram/ml) and avidity index of antibodies to the P falciparum CS protein by ELISA, the geometric mean titre to P falciparum sporozoites by IFAT (1/115 vs 1/1600), and the geometric mean inhibition of sporozoite invasion of hepatoma cells in vitro (37.6 vs 90.3%). For R32NS1(81) MPL/CWS is superior to aluminum hydroxide as an adjuvant, and the data support the evaluation of this complex as an adjuvant for other vaccines.

As part of a study of potential vaccines against malaria, the protective efficacy of sporozoite subunit vaccines was determined by using the Plasmodium berghei murine malaria model. Mice were immunized with recombinant DNA-produced or synthetic peptide-carrier subunit vaccines derived from the repetitive epitopes of the Plasmodium berghei circumsporozoite gene, or with radiation-attenuated sporozoites. Immunization with subunit vaccines elicited humoral responses that were equivalent to or greater than those elicited by irradiated sporozoites, yet the protection against sporozoite challenge induced by either of the subunit vaccines was far less than that achieved by immunization with attenuated sporozoites. Passive and adoptive transfer studies demonstrated that subunit vaccines elicited predominantly antibody-mediated protection that was easily overcome whereas irradiated sporozoites induced potent cell-mediated immunity that protected against high challenge doses of sporozoites. These studies indicate that new strategies designed to induce cellular immunity will be required for efficacious sporozoite vaccines.

Human retroplacental serum (RPS) containing polyamine oxidase inhibited the growth of the Camp strain of Plasmodium falciparum in vitro as assayed by the parasite's decreased incorporation of 3H-hypoxanthine. Inhibition was dose-dependent on the concentrations of serum polyamine oxidase and added polyamines. Almost complete inhibition was seen in 96-hr asynchronous cultures containing 10% RPS and in those containing 1.2% RPS plus 50 microM polyamine. Subtle morphologic changes in mature stages and decreased numbers of new rings were associated with inhibition seen in 19-hr synchronous cultures initiated at the trophozoite stage. These incubation times were longer than in previous reports showing inhibition of malaria parasites by bovine polyamine oxidase but not by human polyamine oxidase. Macrophages contain polyamine oxidase, the reaction products of which are known to be similar to those of RPS polyamine oxidase but different from those of bovine polyamine oxidase. It remains to be determined whether human polyamine oxidase, acting upon ubiquitous polyamines, contributes to host defenses against malaria.