Characterization of humoral and cell-mediated immunity induced by mRNA vaccines expressing influenza hemagglutinin stem and nucleoprotein in mice and nonhuman primates.

In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.

Transplacental Antibody Transfer of Respiratory Syncytial Virus Specific IgG in Non-Human Primate Mother-Infant Pairs.

One approach to protect new-borns against respiratory syncytial virus (RSV) is to vaccinate pregnant women in the last trimester of pregnancy. The boosting of circulating antibodies which can be transferred to the foetus would offer immune protection against the virus and ultimately the disease. Since non-human primates (NHPs) have similar reproductive anatomy, physiology, and antibody architecture and kinetics to humans, we utilized this preclinical species to evaluate maternal immunization (MI) using an RSV F subunit vaccine. Three species of NHPs known for their ability to be infected with human RSV in experimental challenge studies were tested for RSV-specific antibodies. African green monkeys had the highest overall antibody levels of the old-world monkeys evaluated and they gave birth to offspring with anti-RSV titers that were proportional to their mother. These higher overall antibody levels are associated with greater durability found in their offspring. Immunization of RSV seropositive AGMs during late pregnancy boosts RSV titers, which consequentially results in significantly higher titers in the vaccinated new-borns compared to the new-borns of unvaccinated mothers. These findings, accomplished in small treatment group sizes, demonstrate a model that provides an efficient, resource sparing and translatable preclinical in vivo system for evaluating vaccine candidates for maternal immunization.

Modified mRNA/lipid nanoparticle-based vaccines expressing respiratory syncytial virus F protein variants are immunogenic and protective in rodent models of RSV infection.

The RSV Fusion (F) protein is a target for neutralizing antibody responses and is a focus for vaccine discovery; however, the process of RSV entry requires F to adopt a metastable prefusion form and transition to a more stable postfusion form, which displays less potent neutralizing epitopes. mRNA vaccines encode antigens that are translated by host cells following vaccination, which may allow conformational transitions similar to those observed during natural infection to occur. Here we evaluate a panel of chemically modified mRNA vaccines expressing different forms of the RSV F protein, including secreted, membrane associated, prefusion-stabilized, and non-stabilized structures, for conformation, immunogenicity, protection, and safety in rodent models. Vaccination with mRNA encoding native RSV F elicited antibody responses to both prefusion- and postfusion-specific epitopes, suggesting that this antigen may adopt both conformations in vivo. Incorporating prefusion stabilizing mutations further shifts the immune response toward prefusion-specific epitopes, but does not impact neutralizing antibody titer. mRNA vaccine candidates expressing either prefusion stabilized or native forms of RSV F protein elicit robust neutralizing antibody responses in both mice and cotton rats, similar to levels observed with a comparable dose of adjuvanted prefusion stabilized RSV F protein. In contrast to the protein subunit vaccine, mRNA-based vaccines elicited robust CD4+ and CD8+ T-cell responses in mice, highlighting a potential advantage of the technology for vaccines requiring a cellular immune response for efficacy.

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation.

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo-trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR-FTIR) reduction in ß-sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α-helix content increased by 7% (CD) to 13% (ATIR-FTIR). Maintenance of a native-like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

Isolation of an in Vitro Affinity-Matured, Thermostable "Headless" HA Stem Fragment That Binds Broadly Neutralizing Antibodies with High Affinity.

The surface glycoprotein hemagglutinin (HA) of influenza virus is the primary target for the design of an effective universal influenza vaccine as it is capable of eliciting broadly cross-reactive antibodies against different HA subtypes. Several monoclonal antibodies targeting the stem region of HA that are able to neutralize various subtypes of influenza virus have been isolated in the recent past. Designing a stable, HA stem immunogen that attains a native-like conformation and can elicit such antibodies has been a challenge. We describe the affinity maturation of a previously designed stem immunogen (H1HA6) by random mutagenesis, followed by selection using yeast surface display. The affinity-matured mutant protein (H1HA6P2), upon bacterial expression, attained a stable, native-like, trimeric conformation without any heterologous trimerization motif and showed a significant improvement in thermal stability and binding to several stem specific, conformation-sensitive, broadly neutralizing antibodies (bnAbs) relative to H1HA6. These results point to an effective strategy for the design of stabilized HA stem immunogens that can be tested for their protective ability.

Structure-based Design of Cyclically Permuted HIV-1 gp120 Trimers That Elicit Neutralizing Antibodies.

A major goal for HIV-1 vaccine development is an ability to elicit strong and durable broadly neutralizing antibody (bNAb) responses. The trimeric envelope glycoprotein (Env) spikes on HIV-1 are known to contain multiple epitopes that are susceptible to bNAbs isolated from infected individuals. Nonetheless, all trimeric and monomeric Env immunogens designed to date have failed to elicit such antibodies. We report the structure-guided design of HIV-1 cyclically permuted gp120 that forms homogeneous, stable trimers, and displays enhanced binding to multiple bNAbs, including VRC01, VRC03, VRC-PG04, PGT128, and the quaternary epitope-specific bNAbs PGT145 and PGDM1400. Constructs that were cyclically permuted in the V1 loop region and contained an N-terminal trimerization domain to stabilize V1V2-mediated quaternary interactions, showed the highest homogeneity and the best antigenic characteristics. In guinea pigs, a DNA prime-protein boost regimen with these new gp120 trimer immunogens elicited potent neutralizing antibody responses against highly sensitive Tier 1A isolates and weaker neutralizing antibody responses with an average titer of about 115 against a panel of heterologous Tier 2 isolates. A modest fraction of the Tier 2 virus neutralizing activity appeared to target the CD4 binding site on gp120. These results suggest that cyclically permuted HIV-1 gp120 trimers represent a viable platform in which further modifications may be made to eventually achieve protective bNAb responses.

Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein.

Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7.

Hemagglutinin Sequence Conservation Guided Stem Immunogen Design from Influenza A H3 Subtype.

Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential "universal" vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, "isoleucine-zipper", or "foldon". These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection.

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.

Enhanced production of Chikungunya virus-like particles using a high-pH adapted spodoptera frugiperda insect cell line.

Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models and are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable and robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in standard culture media (pH 6.2-6.4), properly processed Chikungunya structural proteins were detected and assembled capsids were observed. However, an increase in culture pH to 6.6-6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability and Sf21 culture, medium modifications were made and a novel insect cell variant (SfBasic) was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0-7.2 and produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line and production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein and VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.

Protection provided by a herpes simplex virus 2 (HSV-2) glycoprotein C and D subunit antigen vaccine against genital HSV-2 infection in HSV-1-seropositive guinea pigs.

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.

Live attenuated herpes simplex virus 2 glycoprotein E deletion mutant as a vaccine candidate defective in neuronal spread.

A herpes simplex virus 2 (HSV-2) glycoprotein E deletion mutant (gE2-del virus) was evaluated as a replication-competent, attenuated live virus vaccine candidate. The gE2-del virus is defective in epithelial cell-to-axon spread and in anterograde transport from the neuron cell body to the axon terminus. In BALB/c and SCID mice, the gE2-del virus caused no death or disease after vaginal, intravascular, or intramuscular inoculation and was 5 orders of magnitude less virulent than wild-type virus when inoculated directly into the brain. No infectious gE2-del virus was recovered from dorsal root ganglia (DRG) after multiple routes of inoculation; however, gE2-del DNA was detected by PCR in lumbosacral DRG at a low copy number in some mice. Importantly, no recurrent vaginal shedding of gE2-del DNA was detected in immunized guinea pigs. Intramuscular immunization outperformed subcutaneous immunization in all parameters evaluated, although individual differences were not significant, and two intramuscular immunizations were more protective than one. Immunized animals had reduced vaginal disease, vaginal titers, DRG infection, recurrent genital lesions, and recurrent vaginal shedding of HSV-2 DNA; however, protection was incomplete. A combined modality immunization using live virus and HSV-2 glycoprotein C and D subunit antigens in guinea pigs did not totally eliminate recurrent lesions or recurrent vaginal shedding of HSV-2 DNA. The gE2-del virus used as an immunotherapeutic vaccine in previously HSV-2-infected guinea pigs greatly reduced the frequency of recurrent genital lesions. Therefore, the gE2-del virus is safe, other than when injected at high titer into the brain, and is efficacious as a prophylactic and immunotherapeutic vaccine.

Immunization with a vaccine combining herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea pigs compared to immunization with gD alone.

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation.

Potent and selective HIV-1 ribonuclease H inhibitors based on a 1-hydroxy-1,8-naphthyridin-2(1H)-one scaffold.

Optimization studies using an HIV RNase H active site inhibitor containing a 1-hydroxy-1,8-naphthyridin-2(1H)-one core identified 4-position substituents that provided several potent and selective inhibitors. The best compound was potent and selective in biochemical assays (IC(50)=0.045 µM, HIV RT RNase H; 13 µM, HIV RT-polymerase; 24 µM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC(50)=0.19 µM) with a modest window with respect to cytotoxicity (CC(50)=3.3 µM).

Biaryl ethers as potent allosteric inhibitors of reverse transcriptase and its key mutant viruses: aryl substituted pyrazole as a surrogate for the pyrazolopyridine motif.

Biaryl ethers were recently reported as potent NNRTIs. Herein, we disclose a detailed effort to modify the previously reported compound 1. We have designed and synthesized a series of novel pyrazole derivatives as a surrogate for pyrazolopyridine motif that were potent inhibitors of HIV-1 RT with nanomolar intrinsic activity on the WT and key mutant enzymes and potent antiviral activity in infected cells.

Biaryl ethers as novel non-nucleoside reverse transcriptase inhibitors with improved potency against key mutant viruses.

Biaryl ethers were recently reported as potent NNRTIs. Herein we disclose a detailed SAR study that led to the biaryl ether 6. This compound possessed excellent potency against WT RT and key clinically observed RT mutants and had an excellent pharmacokinetic profile in rats, dogs, and rhesus macaques. The compound also exhibited a clean safety profile in preclinical safety studies.

Substituted tetrahydroquinolines as potent allosteric inhibitors of reverse transcriptase and its key mutants.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are key elements of multidrug regimens, called HAART (Highly Active Antiretroviral Therapy), that are used to treat HIV-1 infections. Elucidation of the structure-activity relationships of the thiocarbamate moiety of the previous published lead compound 2 provided a series of novel tetrahydroquinoline derivatives as potent inhibitors of HIV-1 RT with nanomolar intrinsic activity on the WT and key mutant enzymes and potent antiviral activity in infected cells. The SAR optimization, mutation profiles, preparation of compounds, and pharmacokinetic profile of compounds are described.

Discovery of 3-{5-[(6-amino-1H-pyrazolo[3,4-b]pyridine-3-yl)methoxy]-2-chlorophenoxy}-5-chlorobenzonitrile (MK-4965): a potent, orally bioavailable HIV-1 non-nucleoside reverse transcriptase inhibitor with improved potency against key mutant viruses.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been shown to be a key component of highly active antiretroviral therapy (HAART). The use of NNRTIs has become part of standard combination antiviral therapies producing clinical outcomes with efficacy comparable to other antiviral regimens. There is, however, a critical issue with the emergence of clinical resistance, and a need has arisen for novel NNRTIs with a broad spectrum of activity against key HIV-1 RT mutations. Using a combination of traditional medicinal chemistry/SAR analyses, crystallography, and molecular modeling, we have designed and synthesized a series of novel, highly potent NNRTIs that possess broad spectrum antiviral activity and good pharmacokinetic profiles. Further refinement of key compounds in this series to optimize physical properties and pharmacokinetics has resulted in the identification of 8e (MK-4965), which has high levels of potency against wild-type and key mutant viruses, excellent oral bioavailability and overall pharmacokinetics, and a clean ancillary profile.

The design and synthesis of diaryl ether second generation HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) with enhanced potency versus key clinical mutations.

Using a combination of traditional Medicinal Chemistry/SAR analysis, crystallography, and molecular modeling, we have designed and synthesized a series of novel, highly potent NNRTIs that possess broad antiviral activity against a number of key clinical mutations.

Two distinct Moloney murine leukemia virus RNAs produced from a single locus dimerize at random.

Two genetically distinct retroviral RNAs can be co-packaged if the RNAs are co-expressed in virion producing cells. For Moloney murine leukemia virus (MLV), co-packaged RNAs are not randomly selected from among all packaging-competent RNAs, but instead primarily associate as homodimers. Here, we tested the hypothesis that the distance between proviral templates might hinder RNA heterodimerization, thus generating the observed preferential homodimerization of co-expressed MLV RNAs. To do this, two genetically distinct RNAs were co-expressed from a single locus and the proportions of hetero- and homodimeric virion RNAs were determined. Unlike RNAs transcribed from two different templates, RNAs transcribed from a single locus dimerized at random. Additionally, in vitro transcription experiments suggested that MLV RNA dimerization can occur more efficiently for longer RNAs during transcription than post-synthesis. Together, these findings show that MLV RNA dimer-partner selection likely occurs either co-transcriptionally or within a pool of transcripts near the proviral template.