IgG2 Antibodies against a Clinical Grade Plasmodium falciparum CSP Vaccine Antigen Associate with Protection against Transgenic Sporozoite Challenge in Mice

The availability of a highly purified and well characterized circumsporozoite protein (CSP) is essential to improve upon the partial success of recombinant CSP-based malaria vaccine candidates. Soluble, near full-length, Plasmodium falciparum CSP vaccine antigen (CS/D) was produced in E. coli under bio-production conditions that comply with current Good Manufacturing Practices (cGMP). A mouse immunogenicity study was conducted using a stable oil-in-water emulsion (SE) of CS/D in combination with the Toll-Like Receptor 4 (TLR4) agonist Glucopyranosyl Lipid A (GLA/SE), or one of two TLR7/8 agonists: R848 (un-conjugated) or 3M-051 (covalently conjugated). Compared to Alum and SE, GLA/SE induced higher CS/D specific antibody response in Balb/c mice. Subclass analysis showed higher IgG2:IgG1 ratio of GLA/SE induced antibodies as compared to Alum and SE. TLR synergy was not observed when soluble R848 was mixed with GLA/SE. Antibody response of 3M051 formulations in Balb/c was similar to GLA/SE, except for the higher IgG2:IgG1 ratio and a trend towards higher T cell responses in 3M051 containing groups. However, no synergistic enhancement of antibody and T cell response was evident when 3M051 conjugate was mixed with GLA/SE. In C57Bl/6 mice, CS/D adjuvanted with 3M051/SE or GLA/SE induced higher CSP repeat specific titers compared to SE. While, 3M051 induced antibodies had high IgG2c:IgG1 ratio, GLA/SE promoted high levels of both IgG1 and IgG2c. GLA/SE also induced more potent T-cell responses compared to SE in two independent C57/BL6 vaccination studies, suggesting a balanced and productive TH1/TH2 response. GLA and 3M-051 similarly enhanced the protective efficacy of CS/D against challenge with a transgenic P. berghei parasite and most importantly, high levels of cytophilic IgG2 antibodies were associated with protection in this model. Our data indicated that the cGMP-grade, soluble CS/D antigen combined with the TLR4-containing adjuvant GLA/SE warrants further evaluation for protective responses in humans

Head-to-Head Comparison of Soluble vs. Qβ VLP Circumsporozoite Protein Vaccines Reveals Selective Enhancement of NANP Repeat Responses.

Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage Qβ VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to Qβ and the CSP-Qβ vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the Qβ-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by Qβ presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the Qβ-CSP vaccine. The data presented here pave the way for further improvement in the Qβ conjugation chemistry and evaluation of both the Qβ-CSP and soluble CSP vaccines in the non-human primate model

Qβ-CSP <i>vs</i>. CSP in Montanide.

<p>Groups of 6 mice were vaccinated thrice with 2.5, 1 and 0.1 μg CSP or Qβ-CSP in Montanide. A, B show the individual data points and mean±SEM titers against full-length protein (A) and NANP repeat peptide (B) 2 weeks post 3^rd vaccination (2WP3). **** (p<0.0001 for ANOVA followed by Tukey’s multiple comparisons test); red data points correspond to mice protected 14 days post challenge and numbers (blue) were protected out of 6. C, Immunofluorescence image of methanol fixed sporozoites stained with 1:2500 dilution of anti-CSP pool (left) or Qβ-CSP serum pool (right) for the 1 μg dose groups. D, E show IgG1, IgG2b and IgG2c levels measured by Luminex and expressed as median fluorescence intensity (MFI) at 1:1000 serum dilution against the NANP peptide (D) or the C-term protein (E).</p

Qβ-CSP <i>vs</i>. CSP in SE, GLA/SE and Alum.

<p>Groups of 10 mice received 3 doses, 3 weeks apart of 2.5 μg CSP+SE, Qβ-CSP+SE, CSP+GLA/SE, Qβ-CSP+GLA/SE or Qβ-CSP+Alum. A, B show the mean±SEM of the full-length and NANP-specific ELISA titers at 2 weeks post third vaccination. ** (p<0.01); * (p<0.05); red symbols represent protected mice and number (blue) protected out of 10. C, D, E and F are data from an independent 2-dose study. 15 mice received 2 doses of 2.5 μg CSP+GLA/SE and Qβ-CSP+GLA/SE, three weeks apart. C, D show the mean±SEM of full-length and NANP-specific ELISA titer from the 10 challenged mice at 2 weeks after the second vaccination. Red symbols were protected mice and number represent protected out of 10 (blue). E, F, IgG1, IgG2b and IgG2c responses of 15 mice measured by Luminex and expressed as MFI at 1:2000 dilution against NANP peptide (E) or C-term protein (F).</p

Combined ELISA titer and protection data of CSP <i>vs</i>. Qβ-CSP groups.

<p>Full-length (left) and NANP (right) titers were plotted for individual animals in Montanide, Alum and SE adjuvanted CSP and Qβ-CSP groups. Combined protection data is indicated (blue). Lines are mean±SEM and the P values are for unpaired T test performed on log transformed titers.</p

Qβ-CSP <i>vs</i>. CSP in Alum.

<p>Groups of 10 mice received 2 doses, 3 weeks apart of 2.5 μg CSP+Alum, Qβ-CSP+Alum or Qβ-CSP without an adjuvant. A, B show the mean±SEM of the full-length and NANP-specific ELISA titers at 2 weeks after the second vaccination. **** (p<0.0001); *** (p<0.001); * (p<0.05); red symbols represent protected mice and number (blue) protected out of 10. C, D, show the IgG1, IgG2b and IgG2c responses measured by Luminex and expressed as MFI at 1:1000 dilution against NANP peptide (C) or C-term protein (D).</p

Characterization of Qβ-CSP vaccine.

<p>A, identity of the product bands was confirmed using reducing coomassie blue staining and western blot with polyclonal mouse anti-Qβ and anti-CSP antibodies. Lanes 1, 2, Qβ-CSP; lane 3, unconjugated Qβ; lane 4, unconjugated CSP. B, particle size distribution of Qβ-CSP by dynamic light scatter analysis. C, electron micrograph of the negatively stained Qβ-CSP. D, immunological reactivity of the soluble CSP and Qβ-CSP against CSP-specific mAbs targeting the NANP repeats, the C-term region or epitopes present only on full-length (FL) CSP. Mabs labelled as “Hu” were produced in humanized mice and “Mo” were produced in wild-type mice.</p

Production of the Qβ-CSP vaccine.

<p>A, Outline of the conjugation process. Numbers in parentheses correspond to the respective lane number in Fig 1B. B, CSP and Qβ proteins analyzed by reducing SDS-PAGE followed by coomassie blue staining (left) or western blot using anti-CSP polyclonal mouse antibodies (right). Lane 1, soluble CSP protein; 2, CSP diluted in urea; 3, SATA treated and desalted CSP; 4, Qβ protein; 5, SMPH treated Qβ; 6, desalted Qβ-Malemide; 7, deacetylated and desalted CSP; 8, CSP-Qβ conjugate; 9, desalted CSP-Qβ conjugate (final vaccine); M, molecular weight marker.</p

Transgenic Parasites Stably Expressing Full-Length \u3ci\u3ePlasmodium falciparum\u3c/i\u3e Circumsporozoite Protein as a Model for Vaccine Down- Selection in Mice Using Sterile Protection as an Endpoint

Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations

Immunological responses induced in Balb/c mice by CS/D covalently conjugated to 3M-051.

<p>Groups on 10 Balb/c mice were immunized two doses of 2.5 µg CSP, 2 months apart in the indicated adjuvant. One month post second immunization sera were collected and analyzed. <i>A,</i> ELISA end point titers of mice, measured against CS/D (left) or NANP repeat peptide (right). <i>B,</i> IgG1 (left) and IgG2a (right) subclasses expressed as Luminex mean fluorescence intensities (MFI) at 1∶500 serum dilution. <i>C</i>, Percentage of total Cytokine⁺CD44⁺CD4⁺ T lymphocytes, extracted from vaccinated Balb/c mice and stimulated with CS/D, stained for intracellular IL-2 (left) or IFN-γ (middle) or CD8⁺ cells stimulated with the K^d-restricted peptide and stained for IFN-γ (right). Lines are mean with SEM.</p