Luis A. Brito

Rational design of small molecules as vaccine adjuvants

Small-molecule immune potentiators can be engineered to be potent adjuvants with localized innate immune activation and short in vivo residence times. Better Adjuvants Through Chemistry Vaccine development has come a long way since Jenner first noticed that cowpox protected against smallpox. And yet, many vaccines do not work well alone; adjuvants are included with the vaccine to boost the immune response. Despite the critical role of adjuvants in vaccine efficacy, new adjuvant development has been empirical. Now, Wu et al. report the rational optimization of small-molecule immune potentiators (SMIPs) as adjuvants. These SMIPs were engineered to have limited bioavailability and remain localized, inducing temporally and spatially restricted inflammation. This systematic approach to optimizing adjuvant properties may allow for improved immune responses to vaccines with fewer side effects. Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.

Acceptable levels of endotoxin in vaccine formulations during preclinical research

This brief commentary reviews endotoxin levels of commercial vaccines and puts them into context for the preclinical researcher working in vaccines. Vaccines are not required to adhere to endotoxin levels as outlined in the United States Pharmacopoeia. Vaccine manufacturers have to show that the vaccine is safe and efficacious in clinical trials. Endotoxin limits are typically lot release specifications for most vaccines, but these values are not available to most researchers designing preclinical experiments. The limits outlined are calculated from endotoxin levels found in a variety of vaccine types such as gene vectors, recombinant subunits, polysaccharide, live attenuated, inactivated and toxoid vaccines. It is clear that certain families of vaccines such as toxoids contain much higher levels of endotoxin, where others such as purified recombinant subunits and gene vectors may contain very low levels.

Designing and building the next generation of improved vaccine adjuvants

Vaccine adjuvants interact with the immune system, to increase the potency of vaccine antigens. Many of the adjuvants currently available were developed with little understanding of how they worked. Highly pure recombinant antigens are typically very poorly immunogenic due to a lack of exogenous immune activating components such as nucleic acids, lipids, and cell membrane components. In this review we discuss the role of adjuvants and their role as delivery systems or immune potentiators. We also highlight the need for appropriate delivery of immune potentiators with several delivery system adjuvants such as alum, emulsions, liposomes, and polymeric particles. The challenges faced by vaccinologists to create the next generation of vaccines can be solved in-part by developing a greater understanding of the impact of delivery, and an appreciation of the key role of pharmaceutical sciences.

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 Novartiss 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 9u2009kb 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.

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.

Rapidly produced SAM ® vaccine against H7N9 influenza is immunogenic in mice

The timing of vaccine availability is essential for an effective response to pandemic influenza. In 2009, vaccine became available after the disease peak, and this has motivated the development of next generation vaccine technologies for more rapid responses. The SAM® vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic lipid nanoparticle (LNP). When used to express seasonal influenza hemagglutinin (HA), a SAM vaccine elicited potent immune responses, comparable to those elicited by a licensed influenza subunit vaccine preparation. When the sequences coding for the HA and neuraminidase (NA) genes from the H7N9 influenza outbreak in China were posted on a web-based data sharing system, the combination of rapid and accurate cell-free gene synthesis and SAM vaccine technology allowed the generation of a vaccine candidate in 8 days. Two weeks after the first immunization, mice had measurable hemagglutinin inhibition (HI) and neutralizing antibody titers against the new virus. Two weeks after the second immunization, all mice had HI titers considered protective. If the SAM vaccine platform proves safe, potent, well tolerated and effective in humans, fully synthetic vaccine technologies could provide unparalleled speed of response to stem the initial wave of influenza outbreaks, allowing first availability of a vaccine candidate days after the discovery of a new virus.

Nucleic acid vaccines: prospects for non-viral delivery of mRNA vaccines.

Introduction: Nucleic acid-based vaccines are being developed as a means to combine the positive attributes of both live-attenuated and subunit vaccines. Viral vectors and plasmid DNA vaccines have been extensively evaluated in human clinical trials and have been shown to be safe and immunogenic, although none have been licensed for human use. More recently, mRNA-based vaccine alternatives have emerged and might offer certain advantages over their DNA-based counterparts. Areas covered: This review describes the two main categories of mRNA vaccines: conventional non-amplifying and self-amplifying mRNA. It summarizes the initial clinical proof-of-concept studies and outlines the preclinical testing of the next wave of innovations for the technology. Finally, this review highlights the versatile functionality of the mRNA molecule and introduces opportunities for future improvements in vaccine design. Expert opinion: The prospects for mRNA vaccines are very promising. Like other types of nucleic acid vaccines, mRNA vaccines have the potential to combine the positive attributes of live attenuated vaccines while obviating many potential safety limitations. Although data from initial clinical trials appear encouraging, mRNA vaccines are far from a commercial product. These initial approaches have spurred innovations in vector design, non-viral delivery, large-scale production and purification of mRNA to quickly move the technology forward. Some improvements have already been tested in preclinical models for both prophylactic and therapeutic vaccine targets and have demonstrated their ability to elicit potent and broad immune responses, including functional antibodies, type 1 T helper cells-type T cell responses and cytotoxic T cells. Though the initial barriers for this nucleic acid vaccine approach seem to be overcome, in our opinion, the future and continued success of this approach lies in a more extensive evaluation of the many non-viral delivery systems described in the literature and gaining a better understanding of the mechanism of action to allow rational design of next generation technologies.

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.

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.

Vectored co-delivery of human cytomegalovirus gH and gL proteins elicits potent complement-independent neutralizing antibodies

Human cytomegalovirus (hCMV) is prevalent worldwide with infection generally being asymptomatic. Nevertheless, hCMV infection can lead to significant morbidity and mortality. Primary infection of seronegative women or reactivation/re-infection of seropositive women during pregnancy can result in transmission to the fetus, leading to severe neurological defects. In addition, hCMV is the most common viral infection in immunosuppressed organ transplant recipients and can produce serious complications. Hence, a safe and effective vaccine to prevent hCMV infection is an unmet medical need. Neutralizing antibodies to several hCMV glycoproteins, and complexes thereof, have been identified in individuals following hCMV infection. Interestingly, a portion of the CMV-specific neutralizing antibody responses are directed to epitopes found on glycoprotein complexes but not the individual proteins. Using an alphavirus replicon particle (VRP) vaccine platform, we showed that bicistronic VRPs encoding hCMV gH and gL glycoproteins produce gH/gL complexes in vitro. Furthermore, mice vaccinated with these gH/gL-expressing VRPs produced broadly cross-reactive complement-independent neutralizing antibodies to hCMV. These neutralizing antibody responses were of higher titer than those elicited in mice vaccinated with monocistronic VRPs encoding gH or gL antigens, and they were substantially more potent than those raised by VRPs encoding gB. These findings underscore the utility of co-delivery of glycoprotein components such as gH and gL for eliciting potent, broadly neutralizing immune responses against hCMV, and indicate that the gH/gL complex represents a potential target for future hCMV vaccine development.