Cholesterol-Amino-Phosphate (CAP) Derived Lipid Nanoparticles for Delivery of Self-Amplifying RNA and Restoration of Spermatogenesis in Infertile Mice.

Male infertility caused by genetic mutations is an important type of infertility. Currently, there is no reliable method in the clinic to address this medical need. The emergence of mRNA therapy provides a possible strategy for restoring mutant genes in the reproductive system. However, effective delivery of mRNA to spermatocytes remains a formidable challenge. Here a series of cholesterol-amino-phosphate (CAP) lipids are reported by integrating three bioactive moieties into a geometric structure, which is favorable for mRNA delivery. The results demonstrate that CAP-derived lipid nanoparticles (CAP LNPs) can deliver RNA including traditional mRNA and self-amplifying RNA (saRNA) encoding DNA Meiotic Recombinase 1 (Dmc1) protein in spermatocytes and treat male infertility caused by the Dmc1 gene mutation. Notably, the delivery efficiency of CAP LNPs is significantly higher than that of the MC3 and ALC-0315 LNPs, which is consistent with the design of CAP molecules. More importantly, a single injection of CAP LNPs-saRNA can produce Dmc1 protein for an extended period, which restores the spermatogenesis in the Dmc1 gene knockout mouse model. Overall, this study proves the concept of LNPs for the delivery of mRNA to spermatocytes, which provides a unique method to probe male infertility caused by the genetic mutation.

Progress in vaccine development for infectious diseases-a Keystone Symposia report.

The COVID-19 pandemic has taught us many things, among the most important of which is that vaccines are one of the cornerstones of public health that help make modern longevity possible. While several different vaccines have been successful at stemming the morbidity and mortality associated with various infectious diseases, many pathogens/diseases remain recalcitrant to the development of effective vaccination. Recent advances in vaccine technology, immunology, structural biology, and other fields may yet yield insight that will address these diseases; they may also help improve societies' preparedness for future pandemics. On June 1-4, 2022, experts in vaccinology from academia, industry, and government convened for the Keystone symposium "Progress in Vaccine Development for Infectious Diseases" to discuss state-of-the-art technologies, recent advancements in understanding vaccine-mediated immunity, and new aspects of antigen design to aid vaccine effectiveness.

Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines.

The recent clinical success of multiple mRNA-based SARS-CoV-2 vaccines has proven the potential of RNA formulated in lipid nanoparticles (LNPs) in humans, and products based on base-modified RNA, sequence-optimized RNA, and self-replicating RNAs formulated in LNPs are all in various stages of clinical development. However, much remains to be learned about critical parameters governing the manufacturing and use of LNP-RNA formulations. One important issue that has received limited attention in the literature to date is the identification of optimal storage conditions for LNP-RNA that preserve long-term activity of the formulations. Here, we analyzed the physical structure, in vivo expression characteristics, and functional activity of alphavirus-derived self-replicating RNA (repRNA)-loaded LNPs encoding HIV vaccine antigens following storage in varying temperatures, buffers, and in the presence or absence of cryoprotectants. We found that for lipid nanoparticles with compositions similar to clinically-used LNPs, storage in RNAse-free PBS containing 10% (w/v) sucrose at -20 °C was able to maintain vaccine stability and in vivo potency at a level equivalent to freshly prepared vaccines following 30 days of storage. LNPs loaded with repRNA could also be lyophilized with retention of bioactivity.

Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques.

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting broadly neutralizing antibody responses to CoVs. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein with a two-shot protocol generated potent serum receptor binding domain cross-neutralizing antibody responses to both SARS-CoV-2 and SARS-CoV-1. Furthermore, responses were equally effective against most SARS-CoV-2 variants of concern (VOCs) and some were highly effective against Omicron. This result contrasts with human infection or many two-shot vaccination protocols where responses were typically more SARS-CoV-2 specific and where VOCs were less well neutralized. Structural studies showed that cloned macaque neutralizing antibodies, particularly using a given heavy chain germline gene, recognized a relatively conserved region proximal to the angiotensin converting enzyme 2 receptor binding site (RBS), whereas many frequently elicited human neutralizing antibodies targeted more variable epitopes overlapping the RBS. B cell repertoire differences between humans and macaques appeared to influence the vaccine response. The macaque neutralizing antibodies identified a pan-SARS-related virus epitope region less well targeted by human antibodies that could be exploited in rational vaccine design.

Intranasal vaccination with lipid-conjugated immunogens promotes antigen transmucosal uptake to drive mucosal and systemic immunity.

To combat the HIV epidemic and emerging threats such as SARS-CoV-2, immunization strategies are needed that elicit protection at mucosal portals of pathogen entry. Immunization directly through airway surfaces is effective in driving mucosal immunity, but poor vaccine uptake across the mucus and epithelial lining is a limitation. The major blood protein albumin is constitutively transcytosed bidirectionally across the airway epithelium through interactions with neonatal Fc receptors (FcRn). Exploiting this biology, here, we demonstrate a strategy of "albumin hitchhiking" to promote mucosal immunity using an intranasal vaccine consisting of protein immunogens modified with an amphiphilic albumin-binding polymer-lipid tail, forming amph-proteins. Amph-proteins persisted in the nasal mucosa of mice and nonhuman primates and exhibited increased uptake into the tissue in an FcRn-dependent manner, leading to enhanced germinal center responses in nasal-associated lymphoid tissue. Intranasal immunization with amph-conjugated HIV Env gp120 or SARS-CoV-2 receptor binding domain (RBD) proteins elicited 100- to 1000-fold higher antigen-specific IgG and IgA titers in the serum, upper and lower respiratory mucosa, and distal genitourinary mucosae of mice compared to unmodified protein. Amph-RBD immunization induced high titers of SARS-CoV-2-neutralizing antibodies in serum, nasal washes, and bronchoalveolar lavage. Furthermore, intranasal amph-protein immunization in rhesus macaques elicited 10-fold higher antigen-specific IgG and IgA responses in the serum and nasal mucosa compared to unmodified protein, supporting the translational potential of this approach. These results suggest that using amph-protein vaccines to deliver antigen across mucosal epithelia is a promising strategy to promote mucosal immunity against HIV, SARS-CoV-2, and other infectious diseases.

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

A particulate saponin/TLR agonist vaccine adjuvant alters lymph flow and modulates adaptive immunity.

Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immune-stimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast cell­dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally related adjuvant AS01B in mice, and primed robust germinal center B cell, TFH, and HIV tier 2 neutralizing antibodies in nonhuman primates. Together, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest that SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

Phosphate-mediated coanchoring of RBD immunogens and molecular adjuvants to alum potentiates humoral immunity against SARS-CoV-2.

There is a need for additional rapidly scalable, low-cost vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to achieve global vaccination. Aluminum hydroxide (alum) adjuvant is the most widely available vaccine adjuvant but elicits modest humoral responses. We hypothesized that phosphate-mediated coanchoring of the receptor binding domain (RBD) of SARS-CoV-2 together with molecular adjuvants on alum particles could potentiate humoral immunity by promoting extended vaccine kinetics and codelivery of vaccine components to lymph nodes. Modification of RBD immunogens with phosphoserine (pSer) peptides enabled efficient alum binding and slowed antigen clearance, leading to notable increases in germinal center responses and neutralizing antibody titers in mice. Adding phosphate-containing CpG or saponin adjuvants to pSer-RBD:alum immunizations synergistically enhanced vaccine immunogenicity in mice and rhesus macaques, inducing neutralizing responses against SARS-CoV-2 variants. Thus, phosphate-mediated coanchoring of RBD and molecular adjuvants to alum is an effective strategy to enhance the efficacy of SARS-CoV-2 subunit vaccines.

Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice.

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization.

Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2, and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely, viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non-neutralizing or weakly neutralizing ("off-target") antibodies. Using our recently developed electron microscopy polyclonal epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.