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

Abstract

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.

Fig 1. Production of the Qβ-CSP vaccine.

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.

Fig 2. Characterization of Qβ-CSP vaccine.

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.

Fig 3. Qβ-CSP vs. CSP in Montanide.

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).

Fig 4. Qβ-CSP vs. CSP in Alum.

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).

Fig 5. Qβ-CSP vs. CSP in SE, GLA/SE and Alum.

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).

Fig 6. Combined ELISA titer and protection data of CSP vs. Qβ-CSP groups.

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.