Jeffrey A. Lyon

Blood Stage Malaria Vaccine Eliciting High Antigen-Specific Antibody Concentrations Confers No Protection to Young Children in Western Kenya

Objective The antigen, falciparum malaria protein 1 (FMP1), represents the 42-kDa C-terminal fragment of merozoite surface protein-1 (MSP-1) of the 3D7 clone of P. falciparum. Formulated with AS02 (a proprietary Adjuvant System), it constitutes the FMP1/AS02 candidate malaria vaccine. We evaluated this vaccines safety, immunogenicity, and efficacy in African children. Methods A randomised, double-blind, Phase IIb, comparator-controlled trial.The trial was conducted in 13 field stations of one mile radii within Kombewa Division, Nyanza Province, Western Kenya, an area of holoendemic transmission of P. falciparum. We enrolled 400 children aged 12–47 months in general good health.Children were randomised in a 1∶1 fashion to receive either FMP1/AS02 (50 µg) or Rabipur® rabies vaccine. Vaccinations were administered on a 0, 1, and 2 month schedule. The primary study endpoint was time to first clinical episode of P. falciparum malaria (temperature ≥37.5°C with asexual parasitaemia of ≥50,000 parasites/µL of blood) occurring between 14 days and six months after a third dose. Case detection was both active and passive. Safety and immunogenicity were evaluated for eight months after first immunisations; vaccine efficacy (VE) was measured over a six-month period following third vaccinations. Results 374 of 400 children received all three doses and completed six months of follow-up. FMP1/AS02 had a good safety profile and was well-tolerated but more reactogenic than the comparator. Geometric mean anti-MSP-142 antibody concentrations increased from1.3 µg/mL to 27.3 µg/mL in the FMP1/AS02 recipients, but were unchanged in controls. 97 children in the FMP1/AS02 group and 98 controls had a primary endpoint episode. Overall VE was 5.1% (95% CI: −26% to +28%; p-value = 0.7). Conclusions FMP1/AS02 is not a promising candidate for further development as a monovalent malaria vaccine. Future MSP-142 vaccine development should focus on other formulations and antigen constructs. Trial Registration Clinicaltrials.gov NCT00223990

Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm (“codon harmonization”) for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the “recoded” genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli.

Two approximately 300 kilodalton plasmodium falciparum proteins at the surface membrane of infected erythrocytes.

Two very large Plasmodium falciparum proteins are identified as constituents of the infected erythrocyte membrane. Sera were obtained from Aotus monkeys that had been repeatedly infected with asexual P. falciparum from one of four strains. The capacity of these sera to block in vitro cytoadherence of infected erythrocytes and agglutinate intact infected cells was determined. The sera were also used to immunoprecipitate protein antigens from detergent extracts of 125I-surface labeled or biosynthetically radiolabeled infected erythrocytes. For each serum/antigen combination, precipitation of only one protein correlated with the ability of the serum to interfere with cytoadherence and agglutinate infected cells. This malarial protein, denoted Pf EMP 1 (P. falciparum-erythrocyte-membrane-protein 1) bore strain-specific epitope(s) on the cell surface and displayed size heterogeneity (Mr approximately 220,000-350,000). Pf EMP 1 was strongly labeled by cell-surface radioiodination but was a quantitatively very minor malarial protein. Pf EMP 1 was distinguished by its size, surface accessibility and antigenic properties from a more predominant malarial protein in the same size range (Pf EMP 2) that is under the infected erythrocyte membrane at knobs. Monoclonal antibodies and rabbit antisera raised against Pf EMP 2 were used to show that this size heterogeneous antigen was indistinguishable from the previously described MESA (mature parasite infected erythrocyte surface antigen), identified by precipitation with rabbit antisera raised against the MESA hexapeptide repeats. Antibodies raised against Pf EMP 2/MESA did not precipitate Pf EMP 1. We conclude that Pf EMP 1 is either directly responsible for the cytoadherence phenomenon, or is very closely associated with another as yet unidentified functional molecule. Pf EMP 2/MESA must have a structural property/function that is important under the host cell membrane.

The Clinical-Grade 42-Kilodalton Fragment of Merozoite Surface Protein 1 of Plasmodium falciparum Strain FVO Expressed in Escherichia coli Protects Aotus nancymai against Challenge with Homologous Erythrocytic-Stage Parasites

ABSTRACT A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP142 gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP142 (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP142 were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP142 antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP142, but not the MSP142 protein itself or the EGF-like domain 1 fragment. Soluble MSP142 (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.

Sequence comparison of allelic forms of the Plasmodium falciparum merozoite surface antigen MSA2.

MSA2 is a strain variable blood-stage merozoite surface antigen of Plasmodium falciparum. We have derived the MSA2 nucleotide sequence for four cloned parasite isolates. Comparison with three other published sequences suggests that variation may be limited, and that the architecture of the gene can be conveniently described by segregation into four distinct regions. The N and C terminal regions (Regions 1 and 4) are highly conserved in all seven genes. Six of these seven MSA2 genes can be grouped in a single family, within which variation is largely limited to a region characterized by the presence of tandem repeats (Region 2). We have observed two new forms of repeat in a Gly, Ser, Ala-rich block, and noted the absence of repeat in this block of the CAMP strain. The region downstream of the repeat region (Region 3) is highly conserved within this family. Immunochemical analysis reveals that MSA2 is one of the antigens recognized by immune antibodies eluted from intact merozoites. Regions 2 and 3, expressed as recombinant proteins, are recognized by these antibodies, suggesting that these regions are exposed at the surface of the intact merozoite.

Protection Induced by Plasmodium falciparum MSP142 Is Strain-Specific, Antigen and Adjuvant Dependent, and Correlates with Antibody Responses

Vaccination with Plasmodium falciparum MSP142/complete Freunds adjuvant (FA) followed by MSP142/incomplete FA is the only known regimen that protects Aotus nancymaae monkeys against infection by erythrocytic stage malaria parasites. The role of adjuvant is not defined; however complete FA cannot be used in humans. In rodent models, immunity is strain-specific. We vaccinated Aotus monkeys with the FVO or 3D7 alleles of MSP142 expressed in Escherichia coli or with the FVO allele expressed in baculovirus (bv) combined with complete and incomplete FA, Montanide ISA-720 (ISA-720) or AS02A. Challenge with FVO strain P. falciparum showed that suppression of cumulative day 11 parasitemia was strain-specific and could be induced by E. coli expressed MSP142 in combination with FA or ISA-720 but not with AS02A. The coli42-FVO antigen induced a stronger protective effect than the bv42-FVO antigen, and FA induced a stronger protective effect than ISA-720. ELISA antibody (Ab) responses at day of challenge (DOC) were strain-specific and correlated inversely with c-day 11 parasitemia (r = −0.843). ELISA Ab levels at DOC meeting a titer of at least 115,000 ELISA Ab units identified the vaccinees not requiring treatment (noTx) with a true positive rate of 83.3% and false positive rate of 14.3 %. Correlation between functional growth inhibitory Ab levels (GIA) and cumulative day 11 parasitemia was weaker (r = −0.511), and was not as predictive for a response of noTx. The lowest false positive rate for GIA was 30% when requiring a true positive rate of 83.3%. These inhibition results along with those showing that antigen/FA combinations induced a stronger protective immunity than antigen/ISA-720 or antigen/AS02 combinations are consistent with protection as ascribed to MSP1-specific cytophilic antibodies. Development of an effective MSP142 vaccine against erythrocytic stage P. falciparum infection will depend not only on antigen quality, but also upon the selection of an optimal adjuvant component.

Process development and analysis of liver-stage antigen 1, a preerythrocyte-stage protein-based vaccine for Plasmodium falciparum

ABSTRACT Plasmodium falciparum liver-stage antigen 1 (LSA-1) is expressed solely in infected hepatocytes and is thought to have a role in liver schizogony and merozoite release. Specific humoral, cellular, and cytokine immune responses to LSA-1 are well documented, with epitopes identified that correlate with antibody production, proliferative T-cell responses, or cytokine induction. With the goal of developing a vaccine against this preerythrocyte-stage protein, we undertook the good manufacturing practices (GMP) manufacture of a recombinant LSA-1 construct, LSA-NRC, incorporating the N- and C-terminal regions of the protein and two of the centrally placed 17-amino-acid repeats. To improve the protein yield, a method of codon harmonization was employed to reengineer the gene sequence for expression in Escherichia coli. A 300-liter GMP fermentation produced 8 kg of bacterial cell paste, and a three-step column chromatographic method yielded 8 mg of purified antigen per g of paste. The final bulk protein was >98% pure, demonstrated long-term stability, and contained <0.005 endotoxin units per 50 μg of protein. To accomplish the initial stages of evaluation of this protein as a human-use vaccine against malaria, we immunized rabbits and mice with LSA-NRC in Montanide ISA 720. New Zealand White rabbits and A/J (H-2K) mice produced high-titer antibodies that recognized liver-stage parasites in infected cultured human hepatocytes. Gamma interferon-producing cells, which have been associated with LSA-1-mediated protection, were detected in splenocytes harvested from immunized mice. Finally, sera taken from people living in a region where malaria is holoendemic recognized LSA-NRC by Western blotting.

Genetic Vaccination against Malaria Infection by Intradermal and Epidermal Injections of a Plasmid Containing the Gene Encoding the Plasmodium berghei Circumsporozoite Protein

ABSTRACT The circumsporozoite protein (CSP) from the surface of sporozoite stage Plasmodium sp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encoding Plasmodium berghei CSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced by P. berghei CSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.

Safety and Reactogenicity of an MSP-1 Malaria Vaccine Candidate: A Randomized Phase Ib Dose-Escalation Trial in Kenyan Children

Objective: Our aim was to evaluate the safety, reactogenicity, and immunogenicity of an investigational malaria vaccine. Design: This was an age-stratified phase Ib, double-blind, randomized, controlled, dose-escalation trial. Children were recruited into one of three cohorts (dosage groups) and randomized in 2:1 fashion to receive either the test product or a comparator. Setting: The study was conducted in a rural population in Kombewa Division, western Kenya. Participants: Subjects were 135 children, aged 12–47 mo. Interventions: Subjects received 10, 25, or 50 μg of falciparum malaria protein 1 (FMP1) formulated in 100, 250, and 500 μL, respectively, of AS02A, or they received a comparator (Imovax® rabies vaccine). Outcome Measures: We performed safety and reactogenicity parameters and assessment of adverse events during solicited (7 d) and unsolicited (30 d) periods after each vaccination. Serious adverse events were monitored for 6 mo after the last vaccination. Results: Both vaccines were safe and well tolerated. FMP1/AS02A recipients experienced significantly more pain and injection-site swelling with a dose-effect relationship. Systemic reactogenicity was low at all dose levels. Hemoglobin levels remained stable and similar across arms. Baseline geometric mean titers were comparable in all groups. Anti-FMP1 antibody titers increased in a dose-dependent manner in subjects receiving FMP1/AS02A; no increase in anti-FMP1 titers occurred in subjects who received the comparator. By study end, subjects who received either 25 or 50 μg of FMP1 had similar antibody levels, which remained significantly higher than that of those who received the comparator or 10 μg of FMP1. A longitudinal mixed effects model showed a statistically significant effect of dosage level on immune response (F3,1047 = 10.78, or F3, 995 = 11.22, p < 0.001); however, the comparison of 25 μg and 50 μg recipients indicated no significant difference (F1,1047 = 0.05; p = 0.82). Conclusions: The FMP1/AS02A vaccine was safe and immunogenic in malaria-exposed 12- to 47-mo-old children and the magnitude of immune response of the 25 and 50 μg doses was superior to that of the 10 μg dose.

Specificities of antibodies that inhibit merozoite dispersal from malaria-infected erythrocytes

When malaria schizont-infected erythrocytes are cultured with immune serum, antibodies prevent dispersal of merozoites, resulting in the formation of immune clusters of merozoites (ICM) and inhibition of parasite growth. Antigens recognized by these antibodies were identified by probing two dimensional immunoblots of Plasmodium falciparum antigens with antibodies dissociated from immune complexes present at the surface of merozoites in ICM. Total immune serum recognized 88 of the 135 protein spots detected by colloidal gold staining, but antibodies dissociated from immune complexes recognized only 15 protein spots attributable to no more than eight distinct antigens. Antigens recognized by antibodies that inhibit merozoite dispersal include the precursor to the major merozoite surface antigens (gp195), a 126-kDa serine-repeat antigen (SERA), the 130-kDa protein that appears to bind to glycophorin (GBP130), and the approx. 45-kDa merozoite surface antigen. One other antigen (230/215-kDa doublet) was identified by using antibodies affinity purified from recombinant expression proteins. The identities of the other three antigens (150 kDa, 127 kDa and less than 30 kDa) were not determined. This approach provides a strategy for identifying epitopes accessible at the merozoite surface which may be important components of a multivalent vaccine against blood stages of P. falciparum.