Adam Frederik Sander

Background/Objectives: Post-weaning diarrhea remains a major challenge in pig production worldwide. Enterotoxigenic Escherichia coli (ETEC) encoding fimbriae of the F4 type and producing the heat-stable enterotoxin, STb, are one of the important causes of this disease. The aim of the current study was to evaluate whether vaccination of pregnant sows with a novel capsid virus-like particle (cVLP)-based vaccine against F4 and STb (cVLP-FaeG/cVLP-STb) could enhance performance in piglets born after such vaccinated sows. Methods: A field trial was conducted in a commercial sow-to-finisher pig herd. Thirty-five sows were vaccinated twice with the cVLP-FaeG/cVLP-STb vaccine prior to farrowing, while thirty-five control sows were vaccinated twice with commercial vaccines normally used in the herd. Piglets were followed until eight weeks post-weaning to assess antibody responses, diarrhea and treatment incidences, pathogen shedding, and growth performance. Results: Piglets born from immunized sows receiving the cVLP vaccine showed significantly higher serum antibody levels against ETEC F4 throughout the post-weaning period (p ≤ 0.021). The frequency of pathogen detection was similar between groups, while piglets in the cVLP group exhibited significantly lower diarrhea scores at week 6 (p = 0.047), showed a trend of requiring fewer treatments (p = 0.06) and had significantly higher final body weight (p = 0.048). In addition, the cVLP group showed a significantly greater average daily gain over the study period (p = 0.037). Conclusion: Sow immunization with the cVLP vaccine enhanced passive immune protection of piglets, resulting in reduced antimicrobial treatment 2 weeks post-weaning and improved growth performance.

Specific pathogen‐free (SPF) laboratory mice have in recent years been criticized for their limited bench to bedside translational value, specifically within immunological and inflammatory research, due to their immature immune system caused by restricted pathogen exposure. Pathogenic reintroduction would result in the spread of infections, compromising staff safety and animal welfare and a risk of reducing reproducibility of in vivo studies. Previous studies have shown that preimmunizing SPF mice with inactivated murine pathogens efficiently induces increased levels of CD8 ⁺ effector memory T cells and higher inflammatory responses in a skin inflammation model. However, the method is laborious and carries infection risks if inactivation is improper. Therefore, a simpler and more standardized method is preferred to improve reproducibility and safety. We hypothesized that preimmunizing mice with an AP205 capsid virus‐like particle (cVLP) would be a safe, easy, and effective method to stimulate memory T cells in SPF mice and increase the inflammatory response in an ovalbumin‐induced (OVA) dermatitis model similar to the preimmunization with inactivated pathogens. The preimmunized mice seroconverted to the cVLP antigens, but the preimmunization did not induce higher levels of effector memory T cells compared to vehicle treated mice, nor did it affect the inflammatory response in the skin inflammation model. In conclusion, preimmunization with cVLP was not sufficient to induce a cellular immune response in the mice.

The COVID-19 pandemic has emphasized the potential of mRNA vaccines in fighting pandemics, owing to their rapid development, strong immunogenicity and adaptability. However, a drawback is their dose-limiting reactogenicity and inability to generate durable humoral immunity. Here we introduce a modular nucleotide vaccine platform combining the advantages of genetic and capsid virus-like-particle-based vaccines. This platform allows for the display of various antigens on different capsid virus-like particles, improving the magnitude, quality and longevity of the vaccine-induced immune responses. We applied this technology to enhance the immunogenicity of the Pfs25 antigen. Immunization with lipid-nanoparticle-formulated mRNA encoding Pfs25 capsid virus-like particles resulted in higher and potentially more durable anti-Pfs25 antibody responses, along with enhanced functional activity, compared with an mRNA vaccine encoding soluble Pfs25. By improving both humoral and cellular immune responses, this approach may reduce the dose and number of administrations required for effective protection. As a result, it can improve the feasibility of both DNA- and mRNA-based vaccines targeting pandemic and endemic infectious diseases.

Background: Lawsonia intracellularis (LI) is the agent of proliferative enteropathy in swine, a common disease that affects pigs for up to eight weeks after weaning. Aim: To evaluate the effectiveness of two novel subunit vaccines targeting outer membrane proteins on LI. Methods: The two vaccines included OMP2c.cVLP, where the OMP2c antigen was anchored on the surface of capsid virus-like particles (cVLP); and MBP.INVASc, where antigens were anchored to an MBP fusion protein. Groups of six mice, as proof of concept, and six piglets were immunized with either OMP2c.cVLP, MBP.INVASc., or PBS as a control using a prime-boost regime. Results: Both OMP2c.cVLP and MBP.INVASc subunit vaccines induced strong antigen-specific serum IgG and IgA responses. There were no significant differences in weight gain among the groups. Mild-to-moderate clinical signs of LI infection were observed, but vaccinated groups showed lower inflammatory scores and fewer animals tested positive for bacteria by immunohistochemistry. Although neither vaccine completely prevented clinical signs of LI infection, both effectively reduced inflammation and lowered the pathogen load, thereby mitigating the severity of the disease, particularly the MBP.INVASc vaccine. Conclusions: These findings suggest that both vaccines have the potential for further development and optimization to enhance their protective efficacy against LI infections.

A vaccine protecting against malaria caused by Plasmodium falciparum is urgently needed. The blood-stage invasion complex PCRCR consists of the five malarial proteins PfPTRAMP, PfCSS, PfRipr, PfCyRPA, and PfRH5. As each subcomponent represents an essential and highly conserved antigen, PCRCR is considered a promising vaccine target. Furthermore, antibodies targeting the complex can block red blood cell invasion by the malaria parasite. However, extremely high titers of neutralizing antibodies are needed for this invasion-blocking effect, and a vaccine based on soluble PfRH5 protein has proven insufficient in inducing a protective response in a clinical trial. Here, we present the results of two approaches to increase the neutralizing antibody titers: (A) immunofocusing and (B) increasing the immunogenicity of the antigen via multivalent display on capsid virus-like particles (cVLPs). The immunofocusing strategies included vaccinating with peptides capable of binding the invasion-blocking anti-PfCyRPA monoclonal antibody CyP1.9, as well as removing non-neutralizing epitopes of PfCyRPA through truncation. Vaccination with PfCyRPA coupled to the AP205 cVLP induced nearly two-fold higher IgG responses compared to vaccinating with soluble PfCyRPA protein. Immunofocusing using a linear peptide greatly increased the neutralizing capacity of the anti-PfCyRPA antibodies. However, significantly lower total anti-PfCyRPA titers were achieved using this strategy. Our results underline the potential of a cVLP-based malaria vaccine including full-length PfCyRPA, which could be combined with other leading malaria vaccine antigens presented on cVLPs.

The SARS-CoV-2 pandemic and the emergence of novel virus variants have had a dramatic impact on public health and the world economy, underscoring the need for detailed studies that explore the high efficacy of additional vaccines in animal models. In this study, we confirm the pathogenicity of the SARS-CoV-2/Leiden_008 isolate (GenBank accession number MT705206.1) in K18-hACE2 transgenic mice. Using this isolate, we show that a vaccine consisting of capsid virus-like particles (cVLPs) displaying the receptor-binding domain (RBD) of SARS-CoV-2 (Wuhan strain) induces strong neutralizing antibody responses and sterilizing immunity in K18-hACE2 mice. Furthermore, we demonstrate that vaccination with the RBD-cVLP vaccine protects mice from both a lethal infection and symptomatic disease. Our data also indicate that immunization significantly reduces inflammation and lung pathology associated with severe disease in mice. Additionally, we show that the survival of naïve animals significantly increases when sera from animals vaccinated with RBD-cVLP are passively transferred, prior to a lethal virus dose. Finally, the RBD-cVLP vaccine has a similar antigen composition to the clinical ABNCOV2 vaccine, which has shown non-inferiority to the Comirnaty mRNA vaccine in phase I-III trials. Therefore, our study provides evidence that this vaccine design is highly immunogenic and confers full protection against severe disease in mice.

F4-positive enterotoxigenic Escherichia coli is associated with diarrhea and poor growth outcomes in neonatal and newly weaned piglets and is thus a major economic and welfare burden in the swine industry. Vaccination of sows with F4 fimbriae protects against the neonatal disease via passive transfer of maternal immunity. However, this strategy does not protect against infection post-weaning. Consequently, prevention and treatment methods in weaner pigs heavily rely on the use of antimicrobials. Therefore, in order to reduce antimicrobial consumption, more effective prophylactic alternatives are needed. In this study, we describe the development of a capsid virus-like particle (cVLP)-based vaccine targeting the major F4 fimbriae subunit and adhesion molecule, FaeG, and evaluate its immunogenicity in mice, piglets, and sows. cVLP-display significantly increased systemic and mucosal antibody responses towards the recombinant FaeG antigen in mice models. However, in piglets, the presence of anti-F4 maternally derived antibodies severely inhibited the induction of active humoral responses towards the FaeG antigen. This inhibition could not be overcome, even with the enhanced immunogenicity achieved via cVLP display. However, in sows, intramuscular vaccination with the FaeG.cVLP vaccine was able to generate robust IgG and IgA responses that were comparable with a commercial fimbriae-based vaccine, and which were effectively transferred to piglets via colostrum intake. These results demonstrate that cVLP display has the potential to improve the systemic humoral responses elicited against low-immunogenic antigens in pigs; however, this effect is dependent on the use of antigens, which are not the targets of pre-existing maternal immunity.

Capsid virus-like particles (cVLPs), assembled from viral coat proteins, are used as therapeutic cargo delivery vehicles as well as molecular scaffolds for display of vaccine antigens. A versatile vaccine platform has been developed based on the Acinetobacter phage AP205 cVLP, which has been shown to significantly improve antigen-specific antibody responses. This modular cVLP platform exploits a split-protein (Tag/Catcher) conjugation system to enable high-density, unidirectional antigen display. Accordingly, protein antigens can be independently expressed and quality-checked prior to conjugation to pre-assembled cVLPs. Here, we describe considerations for the design of vaccine antigens with genetically fused split-protein (Tag or Catcher) binding partners and provide protocols for the expression and purification of corresponding Tag- or Catcher-AP205 cVLPs from E.coli. Finally, we describe a generic protocol for the formulation and quality assessment of experimental/pre-clinical AP205 cVLP-based vaccines. Key words Virus-like particles cVLP AP205 Tag Catcher Density ultracentrifugation