Vaccines have been the single most significant advancement in public health, preventing morbidity and mortality in millions of people annually. Vaccine development has traditionally focused on whole organism vaccines, either live attenuated or inactivated vaccines. While successful for many different infectious diseases whole organisms are expensive to produce, require culture of the infectious agent, and have the potential to cause vaccine associated disease in hosts. With advancing technology and a desire to develop safe, cost effective vaccine candidates, the field began to focus on the development of recombinantly expressed antigens known as subunit vaccines. While more tolerable, subunit vaccines tend to be less immunogenic. Attempts have been made to increase immunogenicity with the addition adjuvants, either immunostimulatory molecules or an antigen delivery system that increases immune responses to vaccines. An area of extreme interest has been the application of nanotechnology to vaccine development, which allows for antigens to be expressed on a particulate delivery system. One of the most exciting examples of nanovaccines are rationally designed protein nanoparticles. These nanoparticles use some of the basic tenants of structural biology, biophysical chemistry, and vaccinology to develop protective, safe, and easily manufactured vaccines. Rationally developed nanoparticle vaccines are one of the most promising candidates for the future of vaccine development.

The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion SARS-CoV-2 Spike (S), S1 and RBD. These immunogens induce robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A Spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID 50 > 10,000) following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations, and also show durable and potent neutralization against circulating VoC. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2 blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.

Yersinia pestis, the etiologic agent of plague, is closely related to Yersinia pseudotuberculosis evolutionarily but has a very different mode of infection. The RNA-binding regulatory protein, Hfq, mediates regulation by small RNAs (sRNAs) and is required for virulence of both Y. pestis and Y. pseudotuberculosis. Moreover, Hfq is required for growth of Y. pestis, but not Y. pseudotuberculosis, at 37°C. Together, these observations suggest that sRNAs play important roles in the virulence and survival of Y. pestis, and that regulation by sRNAs may account for some of the differences between Y. pestis and Y. pseudotuberculosis. We have used a deep sequencing approach to identify 31 sRNAs in Y. pestis. The majority of these sRNAs are not conserved outside the Yersiniae. Expression of the sRNAs was confirmed by Northern analysis and we developed deep sequencing approaches to map 5ʹ and 3ʹ ends of many sRNAs simultaneously. Expression of the majority of the sRNAs we identified is dependent upon Hfq. We also observed temperature-dependent effects on the expression of many sRNAs, and differences in expression patterns between Y. pestis and Y. pseudotuberculosis. Thus, our data suggest that regulation by sRNAs plays an important role in the lifestyle switch from flea to mammalian host, and that regulation by sRNAs may contribute to the phenotypic differences between Y. pestis and Y. pseudotuberculosis.

To eliminate the problems associated with the use of extraneous adjuvants we have designed a Self-Assembling Protein Nanoparticle (SAPN) containing epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) (designated FMP014) and portions of the TLR5 agonist flagellin (designated FMP014) as an intrinsic adjuvant. By combining different molar ratios of FMP014 to FMP014 monomers before self-assembly, we generated multiple nanoparticles and investigated their biophysical characteristics, immunogenicity and protective efficacy. Immunization with the construct formulated with the ratio 58:2 of FMP014 to FMP014 had the highest protective efficacy against a challenge with a transgenic P. berghei sporozoite expressing PfCSP. Increasing the proportion of flagellin per particle resulted in an inverse relationship with levels of both antibody titers and protection. The cytokine profiles of the various immunization groups were evaluated and quantitative amounts of the cytokines IL-2, IFN-γ, IL-12/p70 (Th1); IL4, IL5 (Th2); TNF-α, IL1β, IL-6, KC/GRO (pro-inflammatory), and IL-10 (immunomodulatory) were measured. The relationship of the cytokines to each other revealed a strong immunomodulatory effect depending on the proportion of flagellin in the construct. Our results demonstrate that SAPNs with flagellin may be a promising strategy for the development and delivery of a safe vaccine for infectious diseases.