Novel approaches for vaccine development.

Vaccines are critical tools for maintaining global health. Traditional vaccine technologies have been used across a wide range of bacterial and viral pathogens, yet there are a number of examples where they have not been successful, such as for persistent infections, rapidly evolving pathogens with high sequence variability, complex viral antigens, and emerging pathogens. Novel technologies such as nucleic acid and viral vector vaccines offer the potential to revolutionize vaccine development as they are well-suited to address existing technology limitations. In this review, we discuss the current state of RNA vaccines, recombinant adenovirus vector-based vaccines, and advances from biomaterials and engineering that address these important public health challenges.

Clinical Development of mRNA Vaccines: Challenges and Opportunities.

The emergence of safe and effective mRNA platform-based COVID-19 vaccines from the recent pandemic has changed the face of vaccine development. Compared with conventional technologies used historically, mRNA-based vaccines offer a rapid flexible and robust approach to preventing disease caused by transient viral strains such as SAR2-CoV-2 variants of concern and seasonal influenza. Adaptations in the formulation of the mRNA delivery systems such as with lipid nanoparticle delivery (LNP) used in mRNA-1273 and BNT16b2b have enabled this technology to flourish under the urgent collective response and collaborative regulatory understanding derived from COVID-19 vaccine development. The application of mRNA-based therapeutics in other areas holds potential promise including combination vaccines that might deliver protections against multiple infectious diseases. Future studies and further advances in mRNA-based technologies will provide insight into the clinical efficacy and real-world effectiveness of vaccines as well as provisions with respect to the impact of reactogenicity profiles. Overall, the success of mRNA-based COVID-19 vaccines has helped unlock a platform likely to result in many more candidate vaccines entering clinical evaluation to address the unmet medical needs of other diseases including viral respiratory diseases, herpesviruses, and historically challenging vaccine targets such as HIV.

Cytomegalovirus (CMV) Pneumonitis: Cell Tropism, Inflammation, and Immunity.

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing disease mainly in immunocompromised patients or after congenital infection. HCMV infection of the respiratory tract leads to pneumonitis in the immunocompromised host, which is often associated with a bad clinical course. The related mouse cytomegalovirus (MCMV) likewise exhibits a distinct tropism for the lung and thus provides an elegant model to study host-pathogen interaction. Accordingly, fundamental features of cytomegalovirus (CMV) pneumonitis have been discovered in mice that correlate with clinical data obtained from humans. Recent studies have provided insight into MCMV cell tropism and localized inflammation after infection of the respiratory tract. Accordingly, the nodular inflammatory focus (NIF) has been identified as the anatomical correlate of immune control in lungs. Several hematopoietic cells involved in antiviral immunity reside in NIFs and their key effector molecules have been deciphered. Here, we review what has been learned from the mouse model with focus on the microanatomy of infection sites and antiviral immunity in MCMV pneumonitis.

Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses.

Recently, the World Health Organization confirmed 120 new human cases of avian H7N9 influenza in China resulting in 37 deaths, highlighting the concern for a potential pandemic and the need for an effective, safe, and high-speed vaccine production platform. Production speed and scale of mRNA-based vaccines make them ideally suited to impede potential pandemic threats. Here we show that lipid nanoparticle (LNP)-formulated, modified mRNA vaccines, encoding hemagglutinin (HA) proteins of H10N8 (A/Jiangxi-Donghu/346/2013) or H7N9 (A/Anhui/1/2013), generated rapid and robust immune responses in mice, ferrets, and nonhuman primates, as measured by hemagglutination inhibition (HAI) and microneutralization (MN) assays. A single dose of H7N9 mRNA protected mice from a lethal challenge and reduced lung viral titers in ferrets. Interim results from a first-in-human, escalating-dose, phase 1 H10N8 study show very high seroconversion rates, demonstrating robust prophylactic immunity in humans. Adverse events (AEs) were mild or moderate with only a few severe and no serious events. These data show that LNP-formulated, modified mRNA vaccines can induce protective immunogenicity with acceptable tolerability profiles.

Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques.

mRNA vaccines are rapidly emerging as a powerful platform for infectious diseases because they are well tolerated, immunogenic, and scalable and are built on precise but adaptable antigen design. We show that two immunizations of modified non-replicating mRNA encoding influenza H10 hemagglutinin (HA) and encapsulated in lipid nanoparticles (LNP) induce protective HA inhibition titers and H10-specific CD4+ T cell responses after intramuscular or intradermal delivery in rhesus macaques. Administration of LNP/mRNA induced rapid and local infiltration of neutrophils, monocytes, and dendritic cells (DCs) to the site of administration and the draining lymph nodes (LNs). While these cells efficiently internalized LNP, mainly monocytes and DCs translated the mRNA and upregulated key co-stimulatory receptors (CD80 and CD86). This coincided with upregulation of type I IFN-inducible genes, including MX1 and CXCL10. The innate immune activation was transient and resulted in priming of H10-specific CD4+ T cells exclusively in the vaccine-draining LNs. Collectively, this demonstrates that mRNA-based vaccines induce type-I IFN-polarized innate immunity and, when combined with antigen production by antigen-presenting cells, lead to generation of potent vaccine-specific responses.

Induction of Broad-Based Immunity and Protective Efficacy by Self-amplifying mRNA Vaccines Encoding Influenza Virus Hemagglutinin.

UNLABELLED: Seasonal influenza is a vaccine-preventable disease that remains a major health problem worldwide, especially in immunocompromised populations. The impact of influenza disease is even greater when strains drift, and influenza pandemics can result when animal-derived influenza virus strains combine with seasonal strains. In this study, we used the SAM technology and characterized the immunogenicity and efficacy of a self-amplifying mRNA expressing influenza virus hemagglutinin (HA) antigen [SAM(HA)] formulated with a novel oil-in-water cationic nanoemulsion. We demonstrated that SAM(HA) was immunogenic in ferrets and facilitated containment of viral replication in the upper respiratory tract of influenza virus-infected animals. In mice, SAM(HA) induced potent functional neutralizing antibody and cellular immune responses, characterized by HA-specific CD4 T helper 1 and CD8 cytotoxic T cells. Furthermore, mice immunized with SAM(HA) derived from the influenza A virus A/California/7/2009 (H1N1) strain (Cal) were protected from a lethal challenge with the heterologous mouse-adapted A/PR/8/1934 (H1N1) virus strain (PR8). Sera derived from SAM(H1-Cal)-immunized animals were not cross-reactive with the PR8 virus, whereas cross-reactivity was observed for HA-specific CD4 and CD8 T cells. Finally, depletion of T cells demonstrated that T-cell responses were essential in mediating heterologous protection. If the SAM vaccine platform proves safe, well tolerated, and effective in humans, the fully synthetic SAM vaccine technology could provide a rapid response platform to control pandemic influenza. IMPORTANCE: In this study, we describe protective immune responses in mice and ferrets after vaccination with a novel HA-based influenza vaccine. This novel type of vaccine elicits both humoral and cellular immune responses. Although vaccine-specific antibodies are the key players in mediating protection from homologous influenza virus infections, vaccine-specific T cells contribute to the control of heterologous infections. The rapid production capacity and the synthetic origin of the vaccine antigen make the SAM platform particularly exploitable in case of influenza pandemic.

Vaccine adjuvant formulations: a pharmaceutical perspective.

Formulation science is an unappreciated and often overlooked aspect in the field of vaccinology. In this review we highlight key attributes necessary to generate well characterized adjuvant formulations. The relationship between the adjuvant and the antigen impacts the immune responses generated by these complex biopharmaceutical formulations. We will use 5 well established vaccine adjuvant platforms; alum, emulsions, liposomes, PLG, and particulate systems such as ISCOMS in addition to immune stimulatory molecules such as MPL to illustrate that a vaccine formulation is more than a simple mixture of component A and component B. This review identifies the challenges and opportunities of these adjuvant platforms. As antigen and adjuvant formulations increase in complexity having a well characterized robust formulation will be critical to ensuring robust and reproducible results throughout preclinical and clinical studies.

Potent immune responses in rhesus macaques induced by nonviral delivery of a self-amplifying RNA vaccine expressing HIV type 1 envelope with a cationic nanoemulsion.

Self-amplifying messenger RNA (mRNA) of positive-strand RNA viruses are effective vectors for in situ expression of vaccine antigens and have potential as a new vaccine technology platform well suited for global health applications. The SAM vaccine platform is based on a synthetic, self-amplifying mRNA delivered by a nonviral delivery system. The safety and immunogenicity of an HIV SAM vaccine encoding a clade C envelope glycoprotein formulated with a cationic nanoemulsion (CNE) delivery system was evaluated in rhesus macaques. The HIV SAM vaccine induced potent cellular immune responses that were greater in magnitude than those induced by self-amplifying mRNA packaged in a viral replicon particle (VRP) or by a recombinant HIV envelope protein formulated with MF59 adjuvant, anti-envelope binding (including anti-V1V2), and neutralizing antibody responses that exceeded those induced by the VRP vaccine. These studies provide the first evidence in nonhuman primates that HIV vaccination with a relatively low dose (50 µg) of formulated self-amplifying mRNA is safe and immunogenic.

A cationic nanoemulsion for the delivery of next-generation RNA vaccines.

Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.

Nonviral delivery of self-amplifying RNA vaccines.

Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.

Co-composting of invasive Acacia longifolia with pine bark for horticultural use.

The feasibility of commercial-scale co-composting of waste biomass from the control of invasive Acacia species with pine bark waste from the lumber industry, in a blend ratio of 60:40 (v:v), was investigated and compared with previous research on the composting of Acacia without additional feedstock, to determine the potential process and end-product quality benefits of co-composting with bark. Pile temperatures rose rapidly to >70 °C and were maintained at >60 °C for several months. Acacia and bark biomass contained a large fraction of mineralizable organic matter (OM) equivalent to approximately 600 g kg(-1) of initial OM. Bark was more recalcitrant to biodegradation compared with Acacia, which degraded at twice the rate of bark. Therefore, incorporating the bark increased the final amount of compost produced compared with composting Acacia residues without bark. The relatively high C/N ratio of the composting matrix (C/N=56) and NH3 volatilization explained the limited increases in NH4+-N content, whereas concentrations of conservative nutrient elements (e.g. P, K, Ca, Mg, Fe) increased in proportion to OM mineralization, enriching the compost as a nutrient source for horticultural use. Nitrogen concentrations also increased to a small extent, but were much more dynamic and losses, probably associated with N volatilization mechanisms, were difficult to actively control. The physicochemical characteristics of the stabilized end-product, such as pH, electrical conductivity and OM content, were improved with the addition of bark to Acacia biomass, and the final compost characteristics were suitable for use for soil improvement and also as horticultural substrate components.

Valorization of kiwi waste through composting.

Kiwi waste from the calibration process is a major environmental problem of kiwi production due to landfill deposition. This work aims to contribute to the agronomic use of recycled kiwi waste through composting. With this objective, a composting experiment was carried out with kiwi fruit waste mixed with 5%, 10% and 20% (fresh weight) of wheat straw from bundles used to protect kiwifruit trunks from frost, as abulking agent to increase aeration, in the piles 5S, 10S and 20S, respectively. The highest temperatures for piles 5S and 10S were above 60°C, whereas the temperature did not reach 40°C in the pile with the highest straw content (20S) because the aeration increased heat loss in addition to increased C/N ratio of this pile. Also, the amount of organic matter mineralized decreased with increasing amount of straw because of the high C/N ratio of the straw. The highest total N (29.7 g kg-1) and the lowest C/N ratio (13) of the compost with 5% of straw is important from the agricultural point of view to promote N availability. In contrast, the high electrical conductivity (4.6 dS m-1) of this compost increases the risk of salt accumulation in the soil. Our results show that the compost with 10% straw, with high degree of maturation, absence of poor hygiene indicators as coliforms and pathogens as Salmonella sp., high organic matter content and rich in nutrients, together with the adequate compost pH and low electrical conductivity improves compost quality.

Composting for management and resource recovery of invasive Acacia species.

The feasibility of commercial-scale composting of waste biomass from the control of invasive Acacia species was investigated. Pile temperatures exceeded 65ºC for several months, indicating that the composting process was effective at pathogen inactivation and seed destruction. Mineralisation of Acacia biomass was described by a two-component, first-order exponential model; the pool sizes for labile and recalcitrant organic matter (OM) were similar and in the approximate ranges: 360-410 g kg(-1) and 350-390 g kg(-1) of initial OM, respectively. Concentrations of conservative nutrients increased proportionally to OM mineralisation, enriching the compost as an agricultural nutrient source. Nitrogen concentrations also increased, but were more dynamic as nitrogen losses were difficult to control, although we suggest that they may be potentially minimised by restricting the turning frequency. The physicochemical characteristics of the stabilised end-product, and the high OM content and low electrical conductivity (<1.2 dS m(-1)), in particular, were suitable for soil improvement or as substrate components.

Phytochemical screening and antibacterial assessment of two macroalgae Ulva papenfussi and Ulva nematoidea (Chlorophyta) against the bacterium Vibrio parahaemolyticus.

This study was conducted to determine whether the macroalgae Ulva papenfussi and Ulva nematoidea could be alternatives for preventing Litopenaeus vannamei vibriosis caused by the bacterium Vibrio parahaemolyticus. Phytochemical screening was performed on methanolic extracts to qualitatively determine the main groups of bioactive compounds, previous to an in vitro antibacterial test against V. parahaemolitycus. Phenols, polyphenols, flavonoids, and the high presence of carbohydrates were found in both macroalgae. U. papenfussi showed more presence of lipids and alkaloids than U. nematoidea . Macroalgae extracts prepared (v:v) with a 1:1 methanol: dichloromethane solvent was used for the in vitro test using the disc diffusion method (MDD). Filter paper discs impregnated with 1.0, 1.5, 2.0, 3.0, and 4.0 mg of the extracts showed antibacterial activity against V. Parahaemolitycus in a dose-dependent manner in both macroalgae. The inhibition zone varied significantly (p < 0.05) from 8.33 ± 0.12 to 11.41 ± 0.73 mm for 1 to 3 mg of extract levels, respectively. In conclusion, both macroalgae have antibacterial activity in their crude extracts against this bacteria. It is suggested to evaluate it as a feed additive for L. vannamei. This study is the first report on a phytochemical screening and antibacterial activity of these macroalgae against V. parahaemolyticus.

Performance of an interferon-γ release assay-based test for cell-mediated immunity to SARS-CoV-2.

In search for immunological correlates of protection against acute coronavirus disease 2019 (COVID-19) there is a need for high through-put assays for cell-mediated immunity (CMI) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We established an interferon-γ release assay -based test for detection of CMI against SARS-CoV-2 spike (S) or nucleocapsid (NC) peptides. Blood samples obtained from 549 healthy or convalescent individuals were measured for interferon-γ (IFN-γ) production after peptide stimulation using a certified chemiluminescence immunoassay. Test performance was calculated applying cutoff values with the highest Youden indices in receiver-operating-characteristics curve analysis and compared to a commercially available serologic test. Potential confounders and clinical correlates were assessed for all test systems. 522 samples obtained from 378 convalescent in median 298 days after PCR-confirmed SARS-CoV-2 infection and 144 healthy control individuals were included in the final analysis. CMI testing had a sensitivity and specificity of up to 89% and 74% for S peptides and 89% and 91% for NC peptides, respectively. High white blood cell counts correlated negatively with IFN-γ responses but there was no CMI decay in samples obtained up to one year after recovery. Severe clinical symptoms at time of acute infection were associated with higher measures of adaptive immunity and reported hair loss at time of examination. This laboratory-developed test for CMI to SARS-CoV-2 NC peptides exhibits excellent test performance, is suitable for high through-put routine diagnostics, and should be evaluated for clinical outcome prediction in prospective pathogen re-exposure.

Indexing of Left Atrial Volume by Body Surface Area and Height in a Brazilian Population without Previous Heart Disease and with a Normal Heart on Echocardiography. Behavior in Obese and Overweight Patients.

Background: Left atrial (LA) volume indexing for body surface area (BSA) may underestimate LA size in obese and overweight people. Since LA volume is a risk marker for some cardiovascular events, it is suggested that indexing for height would be an alternative more appropriate method. The aims of this study were to find normal and the best cutoff values for LA volume indexed for height in our population. Methods: Echocardiograms from 2018 to 2021 were reviewed and patients without known cardiac disease and completely normal echocardiograms that had the left atrial volume (LAvol) measured by biplane Simpson's method were included. LAvol was indexed by BSA (ml/m2), by height (LAvol/m), by height raised to exponent 2.7 (ml/ m2.7) and by height squared (ml/h2). Results: A total of 545 patients, 50.5 ± 13.4 y., 335 females (61,5%) were analyzed. There were 145 normal weight (26.6%), 215 overweight (39.4%), 154 obese (28.3%) and 31 low weight (5.7%) patients. To establish normal values we included only the normal weight group and considered normal values from 2SD below to 2SD above the mean. Mean and normal values were: LAvol/h 26.0 ±4.5, 17 - 35 ml/m, LAvol/ht2 16 ± 2.8, 10.4 - 21.6 ml/ ht2 and LAvol/ht2.7 11.4 ± 2.2, 7.0 - 15.8 ml/m2.7. The normal LAvol/ht2.7 differed between male and female (11.4 ± 2.4 and 12.8 ± 2.6, p < 0.001). LA diameter, LAvol, LAvol/h, LAvol/h2 and LAvol/ht2.7 increased progressively from low-weight, normal weight, overweight and obese patients (p< 0.0001), but not LAvol/BSA. When indexing LAvol for height, for height2 and for height2.7 20.8%, 22.7% and 21.4% of the obese patients, respectively, were reclassified as enlarged LA, and 7.4%, 8.8% and 8.4% of the overweight patients as well. Using ROC curve analysis, LAvol/h2 had the highest AUC ant the best predictive value to identify LA enlargement and LAvol/BSA the worst one. Conclusions: Normal values for LAvol indexed for height by three different methods are described in normal individuals. We reinforce that LAvol indexation for BSA underestimates LA size in obese and overweight patients and in these groups, specially, indexing for height2 is probably the best method to evaluate LAvol.

Subcutaneous delivery of FGF21 mRNA therapy reverses obesity, insulin resistance, and hepatic steatosis in diet-induced obese mice.

Fibroblast growth factor 21 (FGF21) is a promising therapeutic agent for treatment of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH). We show that therapeutic levels of FGF21 were achieved following subcutaneous (s.c.) administration of mRNA encoding human FGF21 proteins. The efficacy of mRNA was assessed following 2-weeks repeated s.c. dosing in diet-induced obese (DIO), mice which resulted in marked decreases in body weight, plasma insulin levels, and hepatic steatosis. Pharmacokinetic/pharmacodynamic (PK/PD) modelling of several studies in both lean and DIO mice showed that mRNA encoding human proteins provided improved therapeutic coverage over recombinant dosed proteins in vivo. This study is the first example of s.c. mRNA therapy showing pre-clinical efficacy in a disease-relevant model, thus, showing the potential for this modality in the treatment of chronic diseases, including T2D and NASH.

Impact of lipid nanoparticle size on mRNA vaccine immunogenicity.

Lipid nanoparticles (LNP) are effective delivery vehicles for messenger RNA (mRNA) and have shown promise for vaccine applications. Yet there are no published reports detailing how LNP biophysical properties can impact vaccine performance. In our hands, a retrospective analysis of mRNA LNP vaccine in vivo studies revealed a relationship between LNP particle size and immunogenicity in mice using LNPs of various compositions. To further investigate this, we designed a series of studies to systematically change LNP particle size without altering lipid composition and evaluated biophysical properties and immunogenicity of the resulting LNPs. While small diameter LNPs were substantially less immunogenic in mice, all particle sizes tested yielded a robust immune response in non-human primates (NHP).

Inefficient Placental Virus Replication and Absence of Neonatal Cell-Specific Immunity Upon Sars-CoV-2 Infection During Pregnancy.

Pregnant women have been carefully observed during the COVID-19 pandemic, as the pregnancy-specific immune adaptation is known to increase the risk for infections. Recent evidence indicates that even though most pregnant have a mild or asymptomatic course, a severe course of COVID-19 and a higher risk of progression to diseases have also been described, along with a heightened risk for pregnancy complications. Yet, vertical transmission of the virus is rare and the possibility of placental SARS-CoV-2 infection as a prerequisite for vertical transmission requires further studies. We here assessed the severity of COVID-19 and onset of neonatal infections in an observational study of women infected with SARS-CoV-2 during pregnancy. Our placental analyses showed a paucity of SARS-CoV-2 viral expression ex vivo in term placentae under acute infection. No viral placental expression was detectable in convalescent pregnant women. Inoculation of placental explants generated from placentas of non-infected women at birth with SARS-CoV-2 in vitro revealed inefficient SARS-CoV-2 replication in different types of placental tissues, which provides a rationale for the low ex vivo viral expression. We further detected specific SARS-CoV-2 T cell responses in pregnant women within a few days upon infection, which was undetectable in cord blood. Our present findings confirm that vertical transmission of SARS-CoV-2 is rare, likely due to the inefficient virus replication in placental tissues. Despite the predominantly benign course of infection in most mothers and negligible risk of vertical transmission, continuous vigilance on the consequences of COVID-19 during pregnancy is required, since the maternal immune activation in response to the SARS-CoV2 infection may have long-term consequences for children's health.