Lung larva reduction (A) and stunted development (B) in mice immunized with rAs16 formulated with ISA720 on Day 8 after being challenged with 2,500 <i>A</i>. <i>suum</i> eggs.

<p>Lung larvae are presented as the mean ± S.D (N = 15). The asterisks indicate statistically significant differences in larval reduction compared to the PBS or adjuvant control groups (*<i>p</i>< 0.05, ***<i>p</i>< 0.001). The larvae collected from lung Baermann culture were observed under 4x objective lens.</p

Properties of recombinant As14 and As16 proteins expressed in <i>P</i>. <i>pastoris</i> X-33.

<p>(<b>A</b>) SDS-PAGE of purified rAs14 and rAs16 proteins (1 μg each). (<b>B</b>) Western blot probed with sera from mice infected with <i>A</i>. <i>suum</i> eggs (diluted 1:3,000) shows that only rAs16 was recognized, but not rAs14 (all antigens 50 ng). (<b>C</b>) Western blot with anti-rAs16 mouse sera (1:3,000). (<b>D</b>) Western blot with anti-rAs14 mouse sera (1:3,000) (all antigens 50 ng). (<b>E</b>) Sequence comparison between As16 and As14 proteins shows 47% sequence identity and 66% similarity.</p

Native As16 and As14 expressed at the lung larval stage of <i>A</i>. <i>suum</i>.

<p>Western blots with anti-As16 or anti-As14 mouse serum (1:3,000) demonstrate that native As16 and As14 are expressed in L3 larvae collected from the lungs of infected mice, but not in infective eggs of <i>A</i>. <i>suum</i>. The unrelated <i>T</i>. <i>cruzi</i> Tc24 protein (50 ng) was used as a negative control.</p

Mouse immune responses to the immunization of rAs16 and rAs14.

<p><b>(A)</b> Anti-As16 and anti-As14 IgG1and IgG2a titers (Log10) in sera of BALB/c mice immunized with rAs16 and rAs14 formulated with ISA720, as measured by ELISA. The IgG1/IgG2a titer ratio for rAs16 immunization is higher than for the rAs14 immunization (2662:1 and 206:1, respectively). <b>(B)</b> Cytokine profiles (IFN-γ, IL-2, IL-4, IL-5 and IL-10) of BALB/c mice immunized with rAs16 and rAs14 formulated with ISA720. Cytokine levels were determined in supernatants of splenocytes after being re-stimulated with rAs16 or rAs14 (25 μg/ml) for 48 hours. Results are shown as means ± standard deviation (SD) and individual data points for each group (n = 5), **<i>p</i><0.01, NS, non-significant.</p

Protective immunity induced by immunization of mice with rAs16 formulated with different adjuvants (Alhydrogel, MPLA and Addavax).

<p>(<b>A</b>) Lung larval count on Day 8 after challenge with 2,500 <i>A</i>. <i>suum</i> eggs. Values are presented as the mean ± S.D. The asterisks indicate statistically significant differences (*<i>p</i> < 0.05) in lung larval reduction compared to the PBS and adjuvant control groups (n = 15). (<b>B</b>) Anti-As16 IgG1 and IgG2a titers (Log₁₀) in sera from BALB/c mice immunized with rAs16 formulated with different adjuvants as measured by ELISA. Values are shown as means ± S.D and individual data points (n = 15). (<b>C</b>) Cytokine profiles (IL-2, IL-4, IL-5 and IL-10, IL-12, GM-CSF, IFN-γ and TNF-α) of BALB/c mice immunized with rAs16 formulated with different adjuvants. Cytokines detected in supernatants of splenocytes after stimulation with rAs16 (25 μg/ml) for 48 hours. Data are presented as means ± S.D and individual values for each group (n = 5). *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001.</p

DataSheet1_Choice of adjuvant and antigen composition alters the immunogenic profile of a SARS-CoV-2 subunit vaccine.PDF

Introduction: Since their introduction, adjuvanted recombinant subunit vaccines against COVID-19 have played a pivotal role in protecting global populations. Optimizing the immune response’s quality, amplitude, and durability to these vaccines depends on the appropriate adjuvant choice and dose in combination with the selected antigen.Methods: Here, we employed a preclinical mouse model to study the adaptive humoral and cellular immune responses to a SARS-CoV-2 receptor binding domain (RBD) antigen formulated with one of four different immune agonists [GLA, 3M-052, CpG-1826 (CpG), and dmLT], in combination with one of two different immune-stimulating formulations, a stabilized squalene emulsion (SE) or aluminum hydroxide (Alum). Using a weighted desirability index, we established an immunogenicity ranking for each adjuvant in combination with the RBD antigen.Results: We found that formulations of the RBD with Alum in combination with either 3M-052 or CpG led to at least a 2-log increase in serum IgG production and a 1.3- to 2.2-log increase in the number of bone marrow-derived antibody-secreting cells compared to the RBD formulated with Alum without an additional agonist. In contrast, the RBD formulated with SE in combination with 3M-052 or CpG did not elicit an IgG response greater than the unadjuvanted control. Additionally, RBD formulated with 3M-052 or CpG on Alum generated a 0.8- or 1.6-log lower splenocyte IL-5 response (a pro-Th2 marker), respectively, than Alum without an additional agonist. When formulated with 3M-052-Alum, a bivalent vaccine containing the original lineage (Wuhan-Hu-1) and the Delta variant (B.1.617.2) RBD antigens led to a more than 2-log increase in neutralizing antibodies against an Omicron variant (B.1.1.529) pseudovirus in vaccinated animals compared to animals that received the monovalent RBD antigen.Discussion: Our results suggest that optimal immune responses to subunit antigens may be achieved through an orthogonal approach that applies adjuvant formulation, antigen combination, and advances in rational vaccine development techniques.</p