A replicon RNA vaccine can induce durable protective immunity from SARS-CoV-2 in nonhuman primates after neutralizing antibodies have waned.

The global SARS-CoV-2 pandemic prompted rapid development of COVID-19 vaccines. Although several vaccines have received emergency approval through various public health agencies, the SARS-CoV-2 pandemic continues. Emergent variants of concern, waning immunity in the vaccinated, evidence that vaccines may not prevent transmission and inequity in vaccine distribution have driven continued development of vaccines against SARS-CoV-2 to address these public health needs. In this report, we evaluated a novel self-amplifying replicon RNA vaccine against SARS-CoV-2 in a pigtail macaque model of COVID-19 disease. We found that this vaccine elicited strong binding and neutralizing antibody responses against homologous virus. We also observed broad binding antibody against heterologous contemporary and ancestral strains, but neutralizing antibody responses were primarily targeted to the vaccine-homologous strain. While binding antibody responses were sustained, neutralizing antibody waned to undetectable levels in some animals after six months but were rapidly recalled and conferred protection from disease when the animals were challenged 7 months after vaccination as evident by reduced viral replication and pathology in the lower respiratory tract, reduced viral shedding in the nasal cavity and lower concentrations of pro-inflammatory cytokines in the lung. Cumulatively, our data demonstrate in pigtail macaques that a self-amplifying replicon RNA vaccine can elicit durable and protective immunity to SARS-CoV-2 infection. Furthermore, these data provide evidence that this vaccine can provide durable protective efficacy and reduce viral shedding even after neutralizing antibody responses have waned to undetectable levels.

Evaluation of repRNA vaccine for induction and in utero transfer of maternal antibodies in a pregnant rabbit model.

Mother-to-child transmission is a major route for infections in newborns. Vaccination in mothers to leverage the maternal immune system is a promising approach to vertically transfer protective immunity. During infectious disease outbreaks, such as the 2016 Zika virus (ZIKV) outbreak, rapid availability of vaccines can prove critical in reducing widespread disease burden. The recent successes of mRNA vaccines support their evaluation in pregnant animal models to justify their use in neonatal settings. Here we evaluated immunogenicity of self-amplifying replicon (repRNA) vaccines, delivered with our clinical-stage LION nanoparticle formulation, in pregnant rabbits using ZIKV and HIV-1 as model disease targets. We showed that LION/repRNA vaccines induced robust antigen-specific antibody responses in adult pregnant rabbits that passively transferred to newborn kits in utero. Using a matrixed study design, we further elucidate the effect of vaccination in kits on the presence of pre-existing maternal antibodies. Our findings showed that timing of maternal vaccination is critical in maximizing in utero antibody transfer, and subsequent vaccination in newborns maintained elevated antibody levels compared with no vaccination. Overall, our results support further development of the LION/repRNA vaccine platform for maternal and neonatal settings.

A localizing nanocarrier formulation enables multi-target immune responses to multivalent replicating RNA with limited systemic inflammation.

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.

Development of LepReact, a defined skin test for paucibacillary leprosy and low-level M. leprae infection.

The persistence of new leprosy cases in endemic areas such as India, Brazil, Bangladesh, and the Philippines has encouraged studies of chemoprophylaxis among contacts of patients. Epidemiological screening tools to enable early detection of infected individuals in endemic populations would be critical to target individuals most in need of intervention. Despite decades of attempts, however, there still are no tests available for the early detection of low-level infection with Mycobacterium leprae. In this report, we describe the development of a leprosy skin test using M. leprae-specific antigens. We selected the chimeric LID-1 fusion protein, formulated to achieve maximum performance at a minimal dose, as a skin test candidate based on its ability to elicit delayed-type hypersensitivity (DTH) reactions in M. leprae immune guinea pigs in a sensitive and specific manner, i.e., with no cross-reactivity observed with other mycobacterial species. Importantly, evaluations in armadillos indicated that intradermal inoculation of formulated LID-1 could distinguish uninfected from M. leprae-infected animals manifesting with symptoms distinctly similar to the PB presentation of patients. Together, our data provide strong proof-of-concept for developing an antigen-specific skin test to detect low-level M. leprae infection. Such a test could, when applied with appropriate use of chemo- and/or immunoprophylaxis, be instrumental in altering the evolution of clinical disease and M. leprae transmission, thus furthering the objective of zero leprosy.

A Nanostructured Lipid Carrier for Delivery of a Replicating Viral RNA Provides Single, Low-Dose Protection against Zika.

Since the first demonstration of in vivo gene expression from an injected RNA molecule almost two decades ago,1 the field of RNA-based therapeutics is now taking significant strides, with many cancer and infectious disease targets entering clinical trials.2 Critical to this success has been advances in the knowledge and application of delivery formulations. Currently, various lipid nanoparticle (LNP) platforms are at the forefront,3 but the encapsulation approach underpinning LNP formulations offsets the synthetic and rapid-response nature of RNA vaccines.4 Second, limited stability of LNP formulated RNA precludes stockpiling for pandemic readiness.5 Here, we show the development of a two-vialed approach wherein the delivery formulation, a highly stable nanostructured lipid carrier (NLC), can be manufactured and stockpiled separate from the target RNA, which is admixed prior to administration. Furthermore, specific physicochemical modifications to the NLC modulate immune responses, either enhancing or diminishing neutralizing antibody responses. We have combined this approach with a replicating viral RNA (rvRNA) encoding Zika virus (ZIKV) antigens and demonstrated a single dose as low as 10 ng can completely protect mice against a lethal ZIKV challenge, representing what might be the most potent approach to date of any Zika vaccine.

Magnetic Particle Imaging: A Novel in Vivo Imaging Platform for Cancer Detection.

Cancer remains one of the leading causes of death worldwide. Biomedical imaging plays a crucial role in all phases of cancer management. Physicians often need to choose the ideal diagnostic imaging modality for each clinical presentation based on complex trade-offs among spatial resolution, sensitivity, contrast, access, cost, and safety. Magnetic particle imaging (MPI) is an emerging tracer imaging modality that detects superparamagnetic iron oxide (SPIO) nanoparticle tracer with high image contrast (zero tissue background signal), high sensitivity (200 nM Fe) with linear quantitation, and zero signal depth attenuation. MPI is also safe in that it uses safe, in some cases even clinically approved, tracers and no ionizing radiation. The superb contrast, sensitivity, safety, and ability to image anywhere in the body lends MPI great promise for cancer imaging. In this study, we show for the first time the use of MPI for in vivo cancer imaging with systemic tracer administration. Here, long circulating MPI-tailored SPIOs were created and administered intravenously in tumor bearing rats. The tumor was highlighted with tumor-to-background ratio of up to 50. The nanoparticle dynamics in the tumor was also well-appreciated, with initial wash-in on the tumor rim, peak uptake at 6 h, and eventual clearance beyond 48 h. Lastly, we demonstrate the quantitative nature of MPI through compartmental fitting in vivo.

Tuning surface coatings of optimized magnetite nanoparticle tracers for in vivo Magnetic Particle Imaging.

Surface coatings are important components of Magnetic Particle Imaging (MPI) tracers - they preserve their key properties responsible for optimum tracer performance in physiological environments. In vivo, surface coatings form a physical barrier between the hydrophobic SPION cores and the physiological environment, and their design dictates the blood half-life and biodistribution of MPI tracers. Here we show the effect of tuning poly(ethylene glycol) (PEG)-based surface coatings on both in vitro and in vivo (mouse model) MPI performance of SPIONs. Our results showed that varying PEG molecular weight had a profound impact on colloidal stability, characterized using Dynamic Light Scattering (DLS), and the m'(H) response of SPIONs, measured in a 25 kHz/20 mTµ0-1max Magnetic Particle Spectrometer (MPS). Increasing PEG molecular weight from 5 kDa to 20 kDa preserved colloidal stability and m'(H) response of ~25 nm SPIONs - the optimum core diameter for MPI - in serum-rich cell culture medium for up to 24 hours. Furthermore, we compared the in vivo circulation time of SPIONs as a function of hydrodynamic diameter and showed that clustered SPIONs can adversely affect blood half-life; critically, SPIONs with clusters had 5 times shorter blood half-life than individually coated SPIONs. We anticipate that the development of MPI SPION tracers with long blood half-lives have potential not only in vascular imaging applications, but also enable opportunities in molecular targeting and imaging - a critical step towards early cancer detection using the new MPI modality.

Variation of Magnetic Particle Imaging Tracer Performance With Amplitude and Frequency of the Applied Magnetic Field.

The magnetic response of magnetic particle imaging (MPI) tracers varies with the slew rate of the applied magnetic field, as well as with the tracer's average magnetic core size. Currently, 25 kHz and 20 mT/µ0 drive fields are common in MPI, but lower field amplitudes may be necessary for patient safety in future designs. We studied how several different sizes of monodisperse MPI tracers behaved under different drive field amplitude and frequency, using magnetic particle spectrometry and ac hysteresis for drive field conditions at 16, 26, and 40 kHz, with field amplitudes from 5 to 40 mT/µ0. We observed that both field amplitude and frequency can influence the tracer behavior, but that the magnetic behavior is consistent when the slew rate (the product of field amplitude and frequency) is consistent. However, smaller amplitudes provide a correspondingly smaller field of view, sometimes resulting in excitation of a minor hysteresis loop.

Accelerated prime-and-trap vaccine regimen in mice using repRNA-based CSP malaria vaccine.

Malaria, caused by Plasmodium parasites, remains one of the most devastating infectious diseases worldwide, despite control efforts to lower morbidity and mortality. Both advanced candidate vaccines, RTS,S and R21, are subunit (SU) vaccines that target a single Plasmodium falciparum (Pf) pre-erythrocytic (PE) sporozoite (spz) surface protein known as circumsporozoite (CS). These vaccines induce humoral immunity but fail to elicit CD8 + T-cell responses sufficient for long-term protection. In contrast, whole-organism (WO) vaccines, such as Radiation Attenuated Sporozoites (RAS), achieved sterile protection but require a series of intravenous doses administered in multiple clinic visits. Moreover, these WO vaccines must be produced in mosquitos, a burdensome process that severely limits their availability. To reduce reliance on WO while maintaining protection via both antibodies and Trm responses, we have developed an accelerated vaccination regimen that combines two distinct agents in a prime-and-trap strategy. The priming dose is a single dose of self-replicating RNA encoding the full-length P. yoelii CS protein, delivered via an advanced cationic nanocarrier (LIONTM). The trapping dose consists of one dose of WO RAS. Our vaccine induces a strong immune response when administered in an accelerated regimen, i.e., either 5-day or same-day immunization. Additionally, mice after same-day immunization showed a 2-day delay of blood patency with 90% sterile protection against a 3-week spz challenge. The same-day regimen also induced durable 70% sterile protection against a 2-month spz challenge. Our approach presents a clear path to late-stage preclinical and clinical testing of dose-sparing, same-day regimens that can confer sterilizing protection against malaria.

Single dose, dual antigen RNA vaccines protect against lethal Crimean-Congo haemorrhagic fever virus infection in mice.

BACKGROUND: Crimean-Congo Haemorrhagic Fever Virus is a tick-borne bunyavirus prevalent across Asia, Africa, the Middle East, and Europe. The virus causes a non-specific febrile illness which may develop into severe haemorrhagic disease. To date, there are no widely approved therapeutics. Recently, we reported an alphavirus-based replicon RNA vaccine which expresses the CCHFV nucleoprotein (repNP) or glycoprotein precursor (repGPC) and is protective against lethal disease in mice. METHODS: Here, we evaluated engineered GPC constructs to find the minimal enhancing epitope of repGPC and test two RNA vaccine approaches to express multiple antigens in vivo to optimize protective efficacy of our repRNA. FINDINGS: Vaccination with repNP and a construct expressing just the Gc antigen (repGc-FL) resulted in equivalent immunogenicity and protective efficacy compared to original repNP + repGPC vaccination. This vaccine was protective when prepared in either of two vaccine approaches, a mixed synthesis reaction producing two RNAs in a single tube and a single RNA expressing two antigens. INTERPRETATION: Overall, our data illustrate two vaccine approaches to deliver two antigens in a single immunization. Both approaches induced protective immune responses against CCHFV in this model. These approaches support their continued development for this and future vaccine candidates for CCHFV and other vaccines where inclusion of multiple antigens would be optimal. FUNDING: This work was supported by the Intramural Research Program, NIAID/NIH, HDT Bio and MCDC Grant #MCDC2204-011.

Size-Dependent Relaxation Properties of Monodisperse Magnetite Nanoparticles Measured Over Seven Decades of Frequency by AC Susceptometry.

Magnetic relaxation is exploited in innovative biomedical applications of magnetic particles such as magnetic particle imaging (MPI), magnetic fluid hyperthermia, and bio-sensing. Relaxation behavior should be optimized to achieve high performance imaging, efficient heating, and good SNR in bio-sensing. Using two AC susceptometers with overlapping frequency ranges, we have measured the relaxation behavior of a series of monodisperse magnetic particles and demonstrated that this approach is an effective way to probe particle relaxation characteristics from a few Hz to 10 MHz, the frequencies relevant for MPI, hyperthermia, and sensing.

Physicochemical and immunological effects of adjuvant formulations with snake venom antigens for immunization of horses for antivenom production.

Enhancement of antivenom immune responses in horses through adjuvant technology improves antivenom production efficiency, but substantial local reactogenicity associated with some traditional veterinary adjuvants limits their usability. To explore modern adjuvant systems suitable for generating antivenom responses in horses, we first assessed their physicochemical compatibility with Bothrops asper snake venom. Liposome and nanoparticle aluminum adjuvants exhibited changes in particle size and phospholipid content after mixing with venom, whereas squalene emulsion-based adjuvants remained stable. Next, we evaluated serum antibody response magnitude and neutralization capacity in horses immunized with adjuvant-containing Echis ocellatus, Bitis arietans, Naja nigricollis, and Dendroaspis polylepis venom preparations. Whereas all tested adjuvants elicited significant neutralization capacity against the viperid venoms, the greatest antibody responses were generated by a squalene-in-water emulsion, thus representing a promising novel alternative for antivenom production.

Immunogenicity and protection against Mycobacterium avium with a heterologous RNA prime and protein boost vaccine regimen.

Prophylactic efficacy of two different delivery platforms for vaccination against Mycobacterium avium (M. avium) were tested in this study; a subunit and an RNA-based vaccine. The vaccine antigen, ID91, includes four mycobacterial antigens: Rv3619, Rv2389, Rv3478, and Rv1886. We have shown that ID91+GLA-SE is effective against a clinical NTM isolate, M. avium 2-151 smt. Here, we extend these results and show that a heterologous prime/boost strategy with a repRNA-ID91 (replicon RNA) followed by protein ID91+GLA-SE boost is superior to the subunit protein vaccine given as a homologous prime/boost regimen. The repRNA-ID91/ID91+GLA-SE heterologous regimen elicited a higher polyfunctional CD4+ TH1 immune response when compared to the homologous protein prime/boost regimen. More significantly, among all the vaccine regimens tested only repRNA-ID91/ID91+GLA-SE induced IFN-γ and TNF-secreting CD8+ T cells. Furthermore, the repRNA-ID91/ID91+GLA-SE vaccine strategy elicited high systemic proinflammatory cytokine responses and induced strong ID91 and an Ag85B-specific humoral antibody response a pre- and post-challenge with M. avium 2-151 smt. Finally, while all prophylactic prime/boost vaccine regimens elicited a degree of protection in beige mice, the heterologous repRNA-ID91/ID91+GLA-SE vaccine regimen provided greater pulmonary protection than the homologous protein prime/boost regimen. These data indicate that a prophylactic heterologous repRNA-ID91/ID91+GLA-SE vaccine regimen augments immunogenicity and confers protection against M. avium.

Accelerated prime-and-trap vaccine regimen in mice using repRNA-based CSP malaria vaccine.

Malaria, caused by Plasmodium parasites, remains one of the most devastating infectious diseases worldwide, despite control efforts that have lowered morbidity and mortality. The only P. falciparum vaccine candidates to show field efficacy are those targeting the asymptomatic pre-erythrocytic (PE) stages of infection. The subunit (SU) RTS,S/AS01 vaccine, the only licensed malaria vaccine to date, is only modestly effective against clinical malaria. Both RTS,S/AS01 and the SU R21 vaccine candidate target the PE sporozoite (spz) circumsporozoite (CS) protein. These candidates elicit high-titer antibodies that provide short-term protection from disease, but do not induce the liver-resident memory CD8+ T cells (Trm) that confer strong PE immunity and long-term protection. In contrast, whole-organism (WO) vaccines, employing for example radiation-attenuated spz (RAS), elicit both high antibody titers and Trm, and have achieved high levels of sterilizing protection. However, they require multiple intravenous (IV) doses, which must be administered at intervals of several weeks, complicating mass administration in the field. Moreover, the quantities of spz required present production difficulties. To reduce reliance on WO while maintaining protection via both antibodies and Trm responses, we have developed an accelerated vaccination regimen that combines two distinct agents in a prime-and-trap strategy. While the priming dose is a self-replicating RNA encoding P. yoelii CS protein, delivered via an advanced cationic nanocarrier (LION™), the trapping dose consists of WO RAS. This accelerated regime confers sterile protection in the P. yoelii mouse model of malaria. Our approach presents a clear path to late-stage preclinical and clinical testing of dose-sparing, same-day regimens that can confer sterilizing protection against malaria.

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease following challenge with heterologous variants of concern.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late 2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoCs) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second-generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform can be updated to target emergent VoCs, elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

Since 2019, the SARS-CoV-2 virus has spread worldwide and caused hundreds of millions of cases of COVID-19. Vaccines were rapidly developed to protect people from becoming severely ill from the virus and decrease the risk of death. However, new variants ­ such as Alpha, Beta and Omicron ­ have emerged that the vaccines do not work as well against, contributing to the ongoing spread of the virus. One way to overcome this is to create a vaccine that can be quickly and easily updated to target new variants, like the vaccine against influenza. Many of the vaccines made against COVID-19 use a new technology to introduce the RNA sequence of the spike protein on the surface of SARS-CoV-2 into our cells. Once injected, our cells use their own machinery to build the protein, or 'antigen', so the immune system can learn how to recognize and destroy the virus. Here, Hawman et al. have renovated an RNA vaccine they made in 2020 which provides immunity against the original strain of SARS-CoV-2 in monkeys and mice. In the newer versions of the vaccine, the RNA was updated with a sequence that matches the spike protein on the Beta or Alpha variant of the virus. Both the original and updated vaccines were then administered to mice and hamsters to see how well they worked against SARS-CoV-2 infections. The experiment showed that all three vaccines caused the animals to produce antibodies that can neutralize the original, Alpha and Beta strains of the virus. Vaccinated hamsters were then infected with one of the three variants ­ either matched or mismatched to their vaccination ­ to see how much protection each vaccine provided. All the vaccines reduced the amount of virus in the animals after infection and mitigated damage in their lungs. But animals that received a vaccine which corresponded to the SARS-CoV-2 strain they were infected with had slightly better protection. These findings suggest that these vaccines work best when their RNA sequence matches the strain responsible for the infection; however, even non-matched vaccines still provide a decent degree of protection. Furthermore, the data demonstrate that the vaccine platform created by Hawman et al. can be easily updated to target new strains of SARS-CoV-2 that may emerge in the future. Recently, the Beta variant of the vaccine entered clinical trials in the United States (led by HDT Bio) to evaluate whether it can be used as a booster in previously vaccinated individuals as well as unvaccinated participants.

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine.

BACKGROUND: In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. METHODS: We rapidly developed a replicating RNA vaccine expressing the B.1.1.529 spike and evaluated immunogenicity in mice and hamsters. We also challenged hamsters with B.1.1.529 and evaluated whether vaccination could protect against viral shedding and replication within respiratory tissue. FINDINGS: We found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine. INTERPRETATION: Our data suggest that B.1.1.529-targeted vaccines may provide superior protection against B.1.1.529 but pre-existing immunity and timing of boosting may need to be considered for optimum protection. FUNDING: This research was supported in part by the Intramural Research Program, NIAID/NIH, Washington Research Foundation and by grants 27220140006C (JHE), AI100625, AI151698, and AI145296 (MG).

Optimizing magnetite nanoparticles for mass sensitivity in magnetic particle imaging.

PURPOSE: Magnetic particle imaging (MPI), using magnetite nanoparticles (MNPs) as tracer material, shows great promise as a platform for fast tomographic imaging. To date, the magnetic properties of MNPs used in imaging have not been optimized. As nanoparticle magnetism shows strong size dependence, the authors explore how varying MNP size impacts imaging performance in order to determine optimal MNP characteristics for MPI at any driving field frequency f0. METHODS: Monodisperse MNPs of varying size were synthesized and their magnetic properties characterized. Their MPI response was measured experimentally using a custom-built MPI transceiver designed to detect the third harmonic of MNP magnetization. The driving field amplitude H0 = 6 mT micro0(-1) and frequency f0 = 250 kHz were chosen to be suitable for imaging small animals. Experimental results were interpreted using a model of dynamic MNP magnetization that is based on the Langevin theory of superparamagnetism and accounts for sample size distribution and size-dependent magnetic relaxation. RESULTS: The experimental results show a clear variation in the MPI signal intensity as a function of MNP diameter that is in agreement with simulated results. A maximum in the plot of MPI signal vs MNP size indicates there is a particular size that is optimal for the chosen f0. CONCLUSIONS: The authors observed that MNPs 15 nm in diameter generate maximum signal amplitude in MPI experiments at 250 kHz. The authors expect the physical basis for this result, the change in magnetic relaxation with MNP size, will impact MPI under other experimental conditions.

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease and pathology and reduce viral shedding following challenge with heterologous SARS-CoV2 variants of concern.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late-2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoC) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

Physicochemical structure of a polyacrylic acid stabilized nanoparticle alum (nanoalum) adjuvant governs TH1 differentiation of CD4+ T cells.

The growing shift to subunit antigen vaccines underscores the need for adjuvants that can enhance the magnitude and quality of immune response. Aluminum salts or alums are the first adjuvants with a long history of clinical use. Alum predominantly induces T helper 2 (TH2) type immunity in animal models, characterized by antibody production with little to no induction of antigen-specific T cells. The lack of cell-mediated or T helper 1 (TH1) immunity makes alum adjuvants ineffective in mounting durable responses against diseases like tuberculosis, malaria and HIV. Here we show that the clinically approved adjuvant, Alhydrogel, reformulated as a stable nanoparticle (nanoalum) with the anionic polymer polyacrylic acid (PAA) induces structure-dependent TH1 response against the recombinant tuberculosis antigen ID93. We found that PAA adsorption to Alhydrogel was a key parameter affecting nanoalum adjuvanticity. Adsorption depended on various factors, most notably formulation pH, and directly correlated with immunological response in mice, enhancing known hallmarks of a murine TH1 type response: induction of antigen-specific IFN-γ secreting CD4+ T cells and IgG2c subclass of antibodies. Our results demonstrate a correlation between a measurable nanoalum property and immunological response, providing a structural basis to derive a beneficial immunological outcome from a clinically approved adjuvant.

Squalene Emulsion Manufacturing Process Scale-Up for Enhanced Global Pandemic Response.

Squalene emulsions are among the most widely employed vaccine adjuvant formulations. Among the demonstrated benefits of squalene emulsions is the ability to enable vaccine antigen dose sparing, an important consideration for pandemic response. In order to increase pandemic response capabilities, it is desirable to scale up adjuvant manufacturing processes. We describe innovative process enhancements that enabled the scale-up of bulk stable squalene emulsion (SE) manufacturing capacity from a 3000- to 5,000,000-dose batch size. Manufacture of concentrated bulk along with the accompanying viscosity change in the continuous phase resulted in a ≥25-fold process efficiency enhancement. Process streamlining and implementation of single-use biocontainers resulted in reduced space requirements, fewer unit operations, and minimization of cleaning requirements. Emulsion physicochemical characteristics were measured by dynamic light scattering, laser diffraction, and HPLC with charged aerosol detection. The newly developed full-scale process was demonstrated by producing two 5,000,000-dose batches of bulk concentrated SE. A scale-up of adjuvant manufacturing capacity through process innovation enables more efficient production capabilities for pandemic response.