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

Vaccination with Plasmodium falciparum MSP142/complete Freund's 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

Correlation between log(c-day 11 parasitemia) and serologic response in animals vaccinated with FVO strain Ag.

<p>Animals were vaccinated with either <i>E. coli</i> or baculovirus expressed MSP1₄₂ in combination with FA, ISA-720 or AS02A. Growth/Invasion inhibition is against FVO (Panel A) or 3D7 (Panel B) strain targets. ELISA was performed with recombinant FVO (Panel C) or 3D7 (Panel D) strain MSP1₄₂ expressed in baculovirus. Correlations were determined with Pearson's test after testing data distributions for normality.</p

Trial Design and Results for Adjuvant CFA/FA

<p><i>Aotus</i> monkeys vaccinated with Ag in combination with FA. Groups of six animals were vaccinated. Day 11 is c-day 11 parasitemia. Tx is treatment outcome: noTx, no treatment required; TxA treatment for anemia; TxP, treatment for uncontrolled parasitemia. GIA is <i>in vitro</i> growth/invasion inhibition against FVO or 3D7 strain <i>P. falciparum.</i> ELISA Ab units were determined with 3D7 or FVO strain MSP1₄₂ expressed in baculovirus. ELISA Ab titers were determined with 3D7 or FVO strain MSP1₄₂, p19, EGF1 or EGF2 expressed in <i>E. coli</i>, and assays were run only using Ag homologous to the vaccine strain. Some results from regimens 1, 3 and 7 were reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002830#pone.0002830-Darko1" target="_blank">[24]</a>. n/d is not done. ^*, no sample available.</p

Trial Design and Results for Adjuvant AS02A.

<p><i>Aotus</i> monkeys vaccinated antigens in combination with AS02A. Groups of six animals were vaccinated. Day 11 is c-day 11 parasitemia. Tx is treatment outcome: noTx, no treatment required; TxA treatment for anemia; TxP, treatment for uncontrolled parasitemia. GIA is <i>in vitro</i> growth/invasion inhibition against FVO or 3D7 strain <i>P. falciparum.</i> ELISA Ab units were determined with 3D7 or FVO strain MSP1₄₂ expressed in baculovirus. ELISA Ab titers were determined with 3D7 or FVO strain MSP1₄₂, p19, EGF1 or EGF2 expressed in <i>E. coli</i>, and assays were run only using Ag homologous to the vaccine strain.</p><p>n/d is not done. ^*, no sample available.</p

C-day 11 parasitemias for regimens 3, 7, 8, and 9 grouped by adjuvant and antigen.

<p>Open circles, individual responses; filled circles, means; bar is the 95% confidence interval for the mean. Panel A compares grouped log(c-day 11 parasitemia) levels. Panel B shows the estimated marginal means from the GLM used to analyze the responses.</p

Individual value plots showing strain-specific Ab responses by ELISA for vaccine recipients.

<p>Responses are grouped by vaccine strain received and then by the Ag strain use for ELISA. Panel A includes all 3D7 strain vaccine recipients in the trial. Panel B includes all FVO strain vaccine recipients in the trial. Connect lines join each individual animal's ELISA Ab response measured with the FVO Ag to that measured with the 3D7 Ag. Both Ag were expressed in baculovirus.</p

Results from ROC graphs for Predicting noTx by Using GIA or ELISA with Ag Expressed in Baculovirus

<p>Responses evaluated were from the twenty-seven animals in vaccine regimens 3, 6, 7, 8 and 9, vaccinated with any FVO Ag in combination with any adjuvant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002830#pone-0002830-t001" target="_blank">Table 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002830#pone-0002830-t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002830#pone-0002830-t003" target="_blank">3</a>). Responses larger than the threshold value predict noTx outcome. %Inhibition is <i>in vitro</i> growth/invasion inhibition against FVO or 3D7 strain <i>P. falciparum.</i> ELISA Ab units were determined with 3D7 or FVO strain MSP1₄₂ expressed in baculovirus.</p

Cumulative Logit Models used to Evaluate the Relationship between Treatment Outcome and Adjuvant and Antigen

<p>Cumulative Logit Models used to Evaluate the Relationship between Treatment Outcome and Adjuvant and Antigen</p

Trial Design and Results for Adjuvant Montanide ISA-720

<p><i>Aotus</i> monkeys vaccinated with one of three MSP1₄₂ antigens in combination with ISA-720. Groups of six animals were vaccinated. Day 11 is c-day 11 parasitemia. Tx is treatment outcome: noTx, no treatment required; TxA treatment for anemia; TxP, treatment for uncontrolled parasitemia. GIA is <i>in vitro</i> growth/invasion inhibition against FVO or 3D7 strain <i>P. falciparum.</i> ELISA Ab units were determined with 3D7 or FVO strain MSP1₄₂ expressed in baculovirus. ELISA Ab titers were determined with 3D7 or FVO strain MSP1₄₂, p19, EGF1 or EGF2 expressed in <i>E. coli</i>, and assays were run only using Ag homologous to the vaccine strain.</p