Safety and immunogenicity of multi-antigen AMA1-based vaccines formulated with CoVaccine HT™ and Montanide ISA 51 in rhesus macaques

<p>Abstract</p> <p>Background</p> <p>Increasing the breadth of the functional antibody response through immunization with <it>Plasmodium falciparum </it>apical membrane antigen 1 (<it>Pf</it>AMA1) multi-allele vaccine formulations has been demonstrated in several rodent and rabbit studies. This study assesses the safety and immunogenicity of three <it>Pf</it>AMA1 Diversity-Covering (DiCo) vaccine candidates formulated as an equimolar mixture (DiCo mix) in CoVaccine HT™ or Montanide ISA 51, as well as that of a <it>Pf</it>AMA1-MSP1₁₉fusion protein formulated in Montanide ISA 51.</p> <p>Methods</p> <p>Vaccine safety in rhesus macaques was monitored by animal behaviour observation and assessment of organ and systemic functions through clinical chemistry and haematology measurements. The immunogenicity of vaccine formulations was assessed by enzyme-linked immunosorbent assays and <it>in vitro </it>parasite growth inhibition assays with three culture-adapted <it>P. falciparum </it>strains.</p> <p>Results</p> <p>These data show that both adjuvants were well tolerated with only transient changes in a few of the chemical and haematological parameters measured. DiCo mix formulated in CoVaccine HT™ proved immunologically and functionally superior to the same candidate formulated in Montanide ISA 51. Immunological data from the fusion protein candidate was however difficult to interpret as four out of six immunized animals were non-responsive for unknown reasons.</p> <p>Conclusions</p> <p>The study highlights the safety and immunological benefits of DiCo mix as a potential human vaccine against blood stage malaria, especially when formulated in CoVaccine HT™, and adds to the accumulating data on the specificity broadening effects of DiCo mix.</p

Generation of Humoral Immune Responses to Multi-Allele PfAMA1 Vaccines; Effect of Adjuvant and Number of Component Alleles on the Breadth of Response

There is increasing interest in multi-allele vaccines to overcome strain-specificity against polymorphic vaccine targets such as Apical Membrane Antigen 1 (AMA1). These have been shown to induce broad inhibitory antibodies in vitro and formed the basis for the design of three Diversity-Covering (DiCo) proteins with similar immunological effects. The antibodies produced are to epitopes that are shared between vaccine alleles and theoretically, increasing the number of component AMA1 alleles is expected to broaden the antibody response. A plateau effect could however impose a limit on the number of alleles needed to achieve the broadest specificity. Moreover, production cost and the vaccine formulation process would limit the number of component alleles. In this paper, we compare rabbit antibody responses elicited with multi-allele vaccines incorporating seven (three DiCos and four natural AMA1 alleles) and three (DiCo mix) antigens for gains in broadened specificity. We also investigate the effect of three adjuvant platforms on antigen specificity and antibody functionality. Our data confirms a broadened response after immunisation with DiCo mix in all three adjuvants. Higher antibody titres were elicited with either CoVaccine HT™ or Montanide ISA 51, resulting in similar in vitro inhibition (65–82%) of five out of six culture-adapted P. falciparum strains. The antigen binding specificities of elicited antibodies were also similar and independent of the adjuvant used or the number of vaccine component alleles. Thus neither the four extra antigens nor adjuvant had any observable benefits with respect to specificity broadening, although adjuvant choice influenced the absolute antibody levels and thus the extent of parasite inhibition. Our data confirms the feasibility and potential of multi-allele PfAMA1 formulations, and highlights the need for adjuvants with improved antibody potentiation properties for AMA1-based vaccines

Mean % antibody depletion from FVO and 3D7 AMA1-coated plates.

<p>Antibody depletion after competition ELISA, values reported as mean % depletion (95%CI).</p><p>7Ag – vaccine containingDiCo mix + four AMA1 alleles from FVO, HB3, 3D7 and CAMP parasite strains.</p><p>3Ag – vaccine containing DiCo mix.</p><p>NM &DM – competitor antigen mixtures comprising natural AMA1 alleles (FVO, HB3, 3D7, CAMP) and DiCo mix, respectively.</p

Mean % antibody depletion from DiCo 1, 2 and 3-coated plates.

<p>Antibody depletion after competition ELISA, values reported as mean % depletion (95%CI).</p><p>7Ag – vaccine containing DiCo mix + four AMA1 alleles from FVO, HB3, 3D7 and CAMP parasite strains.</p><p>3Ag – vaccine containing DiCo mix.</p><p>NM &DM – competitor antigen mixtures comprising natural AMA1 alleles (FVO, HB3, 3D7, CAMP) and DiCo mix, respectively.</p

Islet macrophages drive islet vascular remodeling and compensatory hyperinsulinemia in the early stages of diabetes

Abstract β-cells respond to peripheral insulin resistance by increasing circulating insulin in early type-2 diabetes (T2D). Islet remodeling supports this compensation but the drivers of this process remain poorly understood. Infiltrating macrophages have been implicated in late stage T2D but relatively little is known on islet resident macrophages, especially in early T2D. We hypothesize that islet resident macrophages contribute to islet vascular remodeling and hyperinsulinemia, the failure of which results in a rapid progression to T2D. Using genetic and diet-induced models of compensatory hyperinsulinemia we show that its depletion significantly compromises islet remodeling in terms of size, vascular density and insulin secretion capacity. Depletion of islet macrophages reduces VEGF-A secretion from both human and mouse islets ex vivo and the impact of reduced VEGF-A functionally translates to delayed re-vascularization upon transplantation in vivo. Hence, we show a new role of islet resident macrophages in the context of early T2D and suggest that there is considerable utility in harnessing islet macrophages to promote islet remodeling and islet insulin secretion capacity. Highlights The compensatory hyperinsulinemic phase of type-2 diabetes is accompanied with significant pancreatic islet remodeling. Bona fide islet resident macrophages are increased during the diabetic compensation phase by largely in situ proliferation. Ablating macrophages severely compromises the islet remodeling process and exacerbates glycemic control in vivo . Mouse and human islet macrophages contribute VEGF-A to the islet environment. Specific removal of islet macrophages delays islet vascularization in compensatory hyperinsulinemic mice

Relationship between ELISA antibody titre and <i>in vitro</i> parasite growth inhibition.

<p>Association of antibody levels with <i>in vitro</i> antibody functionality for three of the four immunisation groups (Gps 2, 3 and 4) is shown for parasite strains whose AMA1 allelic antigens were available for antibody measurement. In order to obtain an optimal estimate of the association, growth inhibition data at all four antibody concentrations tested (6.0, 3.0, 1.5 and 0.75 mg/ml) for each sample were included. Plots are based on a four-parameter logistic function, and each symbol/colour represents individual rabbits in the same immunisation group.</p

Alignment of the protein sequences (aa25-545) of <i>Pf</i>AMA1 antigens used in this study.

<p>DiCo proteins were used to immunise rabbits and as capture antigens in ELISA. Natural allele AMA1 proteins were used to immunize rabbits, and as capture and competitor antigens in (competition) ELISAs. All proteins were produced in <i>Pichia pastoris</i> and are devoid of N-glycosylation sites. These have been replaced with amino acid residues (indicated in red) that occur in AMA1 sequences from other malarial species (N162Q, T288V, S373D, N422D, S423K, N499Q). Residue 162 is unique as it is also a polymorphic residue. Additionally, all sequences contain a point mutation at position 376 (K to R, indicated in orange). This was necessary to prevent protein cleavage by <i>P. pastoris</i> proteases.</p

Growth inhibition of <i>Plasmodium</i> parasites by antibodies elicited with the three DiCo mix vaccines.

<p>Antibodies from all immunisation groups were tested on each of six culture-adapted strains (7G8, CAMP, FCR3, HB3, L32 and NF54) of <i>P. falciparum</i>. Plots represent the mean % inhibition ± SEM for all antibody samples within an immunisation group. Blue filled circles (•) represent Gp 2 (DiCo mix in CoVaccine HT™, n = 8), green open diamonds (◊) represent Gp 3 (DiCo mix in Montanide IMS, n = 8) and red filled squares (▪) represent Gp 4 (DiCo mix in Montanide ISA 51, n = 5).</p