Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.

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A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy. We evaluated the efficacy of a form of P. falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P. falciparum parasites. Significant protection from this otherwise lethal challenge with P. falciparum was observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized. The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial. The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.Many malaria vaccine strategies, including our own, depend on including multiple asexual antigens in order (i) to improve coverage of polymorphisms in field isolates, (ii) to overcome individual nonresponsiveness to some antigens, (iii) to improve vaccine efficacy by eliciting immunity to multiple targets, and (iv) to prevent or delay the evolution of escape mutants. If we include two different antigens in a vaccine combination, ideally, synergy in protection will be induced.The most extensive experience in vaccine trials with New World monkeys has been obtained with the C-terminal 42-kDa portion of merozoite surface protein 1 (MSP1 42 ) (3,10,17,18). Recombinant forms of MSP1 42 have been efficacious against homologous parasite challenges, and MSP1 42 is regarded as a leading asexual vaccine candidate.We have now produced a second antigen, Plasmodium falciparum apical membrane antigen 1 (AMA1) (see reference 13a), and in the present study we test the efficacy of this antigen, both alone and in combination with MSP1 42 , in a vaccine trial with Aotus vociferans monkeys.AMA1 is the subject of intensive vaccine research; at least six of the major malaria vaccine research centers have AMA1 programs. This is based on protection against rodent malaria (Plasmodium chabaudi [1,2,6,20] and Plasmodium yoelii [16]) and nonhuman primate malarias (Plasmodium knowlesi [7] and Plasmodium fragile [5]) by use of purified parasite and recombinant antigens and on the generation in rabbits of an in vitro growth-inhibitory antiserum to an Escherichia coli-expressed recombinant P. falciparum AMA1 (12).However, no evidence exists that vaccine-elicited immunity to a recombinant form of P. falciparum AMA1 will be effective against a P. falciparum challenge in vivo, and establishing this prior to human trials is seen by us as essential, especi...

A blood-stage vaccine based on Plasmodium falciparum merozoite surface protein 3 (MSP3) was tested for efficacy in a primate model. Aotus nancymai monkeys were vaccinated with yeast-expressed MSP3 before a lethal challenge with Plasmodium falciparum parasites. Five of 7 control monkeys had acute infections and required treatment to control parasitemia. Only 1 of 7 monkeys vaccinated with MSP3 required this treatment. The efficacy of the MSP3 vaccination appeared to be comparable to that of MSP1(42), a leading asexual vaccine candidate, in response to which 2 monkeys experienced acute infections. In the MSP3-vaccinated group, protection correlated with prechallenge titers of antibody to MSP3. In the MSP1 and control groups, protection correlated with antibody to MSP3 raised by challenge infection.

Two strains of transgenic mice have been generated that secrete into their milk a malaria vaccine candidate, the 42-kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP142). One strain secretes an MSP142 with an amino acid sequence homologous to that of the FVO parasite line, the other an MSP142 where two putative N-linked glycosylation sites in the FVO sequence have been removed. Both forms of MSP142 were purified from whole milk to greater than 91% homogeneity at high yields.

Hypertriglyceridemia, peripheral insulin resistance, and trunk adiposity are metabolic complications recently recognized in people infected with human immunodeficiency virus (HIV) and treated with highly active antiretroviral therapy (HAART). These complications may respond favorably to exercise training. Using a paired design, we determined whether 16 wk of weight-lifting exercise increased muscle mass and strength and decreased fasting serum triglycerides and adipose tissue mass in 18 HIV-infected men. The resistance exercise regimen consisted of three upper and four lower body exercises done for 1-1.5 h/day, 4 days/wk for 64 sessions. Dual-energy X-ray absorptiometry indicated that exercise training increased whole body lean mass 1.4 kg (P = 0.005) but did not reduce adipose tissue mass (P = NS). Axial proton-magnetic resonance imaging indicated that thigh muscle cross-sectional area increased 5-7 cm(2) (P < 0.005). Muscle strength increased 23-38% (P < 0.0001) on all exercises. Fasting serum triglycerides were decreased at the end of training (281-204 mg/dl; P = 0.02). These findings imply that resistance exercise training-induced muscle hypertrophy may promote triglyceride clearance from the circulation of hypertriglyceridemic HIV-infected men treated with antiviral therapy.

Protection against Plasmodium falciparum can be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria. In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP1 42 ) in Escherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities. Biochemical, biophysical, and immunological characterization confirmed that refolded E. coli MSP1 42 was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refolded E. coli MSP1 42 and tested in vitro in a P. falciparum growth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms of P. falciparum MSP1 42 . However, the inhibitory activity was primarily a consequence of antibodies directed against the C-terminal 19 kDa of MSP1 (MSP1 19 ). Vaccination of nonhuman primates with E. coli MSP1 42 in Freund's adjuvant protected six of seven Aotus monkeys from virulent infection with P. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct, E. coli MSP1 42 , is a viable candidate for human vaccine trials.

Unilateral enucleations were performed on larval, transforming and adult sea lampreys. Following 5 to 11 days survival, the animals were sacrificed and the brains were processed using a modified Fink-Heimer technique. In larvae, contralateral optic projections were found to the posterior one-third of the dorsal thalamus, the pretectum, and the optic tectum. No ipsilateral projections were present in the larvae. In enucleated transforming and adult lampreys, degenerating axons were observed in the optic chiasm and bilaterally in the optic tracts. Retinal efferents projected bilaterally to a lateral neuropil region ("tractus opticus") in the posterior one-half of the dorsal thalamus. Contralaterally, a conspicuous dorsomedial cell group (lateral geniculate nucleus) also received a projection. Contralateral projections to the superficial layers of the pretectum and optic tectum were observed. Ipsilateral retinal projections to the pretectum and optic tectum in transforming and adult lampreys were restricted to a small zone at the ventrolateral margins of the pretectum and tectum. The changes in distribution of retinofugal projections during transformation appear to be occurring at the same time that the eye differentiates into its adult form.