Safety, tolerability, pharmacokinetics, and immunogenicity of the therapeutic monoclonal antibody mAb114 targeting Ebola virus glycoprotein (VRC 608): an open-label phase 1 study.

BACKGROUND: mAb114 is a single monoclonal antibody that targets the receptor-binding domain of Ebola virus glycoprotein, which prevents mortality in rhesus macaques treated after lethal challenge with Zaire ebolavirus. Here we present expedited data from VRC 608, a phase 1 study to evaluate mAb114 safety, tolerability, pharmacokinetics, and immunogenicity. METHODS: In this phase 1, dose-escalation study (VRC 608), conducted at the US National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA), healthy adults aged 18-60 years were sequentially enrolled into three mAb114 dose groups of 5 mg/kg, 25 mg/kg, and 50 mg/kg. The drug was given to participants intravenously over 30 min, and participants were followed for 24 weeks. Participants were only enrolled into increased dosing groups after interim safety assessments. Our primary endpoints were safety and tolerability, with pharmacokinetic and anti-drug antibody assessments as secondary endpoints. We assessed safety and tolerability in all participants who received study drug by monitoring clinical laboratory data and self-report and direct clinician assessment of prespecified infusion-site symptoms 3 days after infusion and systemic symptoms 7 days after infusion. Unsolicited adverse events were recorded for 28 days. Pharmacokinetic and anti-drug antibody assessments were completed in participants with at least 56 days of data. This trial is registered with ClinicalTrials.gov, number NCT03478891, and is active but no longer recruiting. FINDINGS: Between May 16, and Sept 27, 2018, 19 eligible individuals were enrolled. One (5%) participant was not infused because intravenous access was not adequate. Of 18 (95%) remaining participants, three (17%) were assigned to the 5 mg/kg group, five (28%) to the 25 mg/kg group, and ten (55%) to the 50 mg/kg group, each of whom received a single infusion of mAb114 at their assigned dose. All infusions were well tolerated and completed over 30-37 min with no infusion reactions or rate adjustments. All participants who received the study drug completed the safety assessment of local and systemic reactogenicity. No participants reported infusion-site symptoms. Systemic symptoms were all mild and present only in four (22%) of 18 participants across all dosing groups. No unsolicited adverse events occurred related to mAb114 and one serious adverse event occurred that was unrelated to mAb114. mAb114 has linear pharmacokinetics and a half-life of 24·2 days (standard error of measurement 0·2) with no evidence of anti-drug antibody development. INTERPRETATION: mAb114 was well tolerated, showed linear pharmacokinetics, and was easily and rapidly infused, making it an attractive and deployable option for treatment in outbreak settings. FUNDING: Vaccine Research Center, US National Institute of Allergy and Infectious Diseases, and NIH.

Neutralizing Alpha-Toxin Accelerates Healing of Staphylococcus aureus-Infected Wounds in Nondiabetic and Diabetic Mice.

Staphylococcus aureus wound infections delay healing and result in invasive complications such as osteomyelitis, especially in the setting of diabetic foot ulcers. In preclinical animal models of S. aureus skin infection, antibody neutralization of alpha-toxin (AT), an S. aureus-secreted pore-forming cytolytic toxin, reduces disease severity by inhibiting skin necrosis and restoring effective host immune responses. However, whether therapeutic neutralization of alpha-toxin is effective against S. aureus-infected wounds is unclear. Herein, the efficacy of prophylactic treatment with a human neutralizing anti-AT monoclonal antibody (MAb) was evaluated in an S. aureus skin wound infection model in nondiabetic and diabetic mice. In both nondiabetic and diabetic mice, anti-AT MAb treatment decreased wound size and bacterial burden and enhanced reepithelialization and wound resolution compared to control MAb treatment. Anti-AT MAb had distinctive effects on the host immune response, including decreased neutrophil and increased monocyte and macrophage infiltrates in nondiabetic mice and decreased neutrophil extracellular traps (NETs) in diabetic mice. Similar therapeutic efficacy was achieved with an active vaccine targeting AT. Taken together, neutralization of AT had a therapeutic effect against S. aureus-infected wounds in both nondiabetic and diabetic mice that was associated with differential effects on the host immune response.

A single amino acid in the F2 subunit of respiratory syncytial virus fusion protein alters growth and fusogenicity.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in children, especially in infants less than 1 year of age. There are currently no licensed vaccines against RSV. rA2ΔM2-2 is a promising live-attenuated vaccine candidate that is currently being evaluated in the clinic. Attenuation of rA2ΔM2-2 is achieved by a single deletion of the M2-2 gene, which disrupts the balance between viral transcription and replication. Whilst performing a manufacturing feasibility study in a serum-free adapted Vero cell line, differences in growth kinetics and cytopathic effect (CPE) were identified between two rA2ΔM2-2 vaccine candidates. Comparative sequence analysis identified four amino acid differences between the two vaccine viruses. Recombinant rA2ΔM2-2 viruses carrying each of the four amino acid differences identified a K66E mutation in the F2 fragment of the fusion (F) protein as the cause of the growth and CPE differences. Syncytium-formation experiments with RSV F protein carrying mutations at aa 66 suggested that a change in charge at this residue within the F2 fragment can have a significant impact on fusion.

Safety and immunogenicity of a ferritin nanoparticle H2 influenza vaccine in healthy adults: a phase 1 trial.

Currently, licensed seasonal influenza vaccines display variable vaccine effectiveness, and there remains a need for novel vaccine platforms capable of inducing broader responses against viral protein domains conserved among influenza subtypes. We conducted a first-in-human, randomized, open-label, phase 1 clinical trial ( NCT03186781 ) to evaluate a novel ferritin (H2HA-Ferritin) nanoparticle influenza vaccine platform. The H2 subtype has not circulated in humans since 1968. Adults born after 1968 have been exposed to only the H1 subtype of group 1 influenza viruses, which shares a conserved stem with H2. Including both H2-naive and H2-exposed adults in the trial allowed us to evaluate memory responses against the conserved stem domain in the presence or absence of pre-existing responses against the immunodominant HA head domain. Fifty healthy participants 18-70 years of age received H2HA-Ferritin intramuscularly as a single 20-µg dose (n = 5) or a 60-µg dose either twice in a homologous (n = 25) prime-boost regimen or once in a heterologous (n = 20) prime-boost regimen after a matched H2 DNA vaccine prime. The primary objective of this trial was to evaluate the safety and tolerability of H2HA-Ferritin either alone or in prime-boost regimens. The secondary objective was to evaluate antibody responses after vaccination. Both vaccines were safe and well tolerated, with the most common solicited symptom being mild headache after both H2HA-Ferritin (n = 15, 22%) and H2 DNA (n = 5, 25%). Exploratory analyses identified neutralizing antibody responses elicited by the H2HA-Ferritin vaccine in both H2-naive and H2-exposed populations. Furthermore, broadly neutralizing antibody responses against group 1 influenza viruses, including both seasonal H1 and avian H5 subtypes, were induced in the H2-naive population through targeting the HA stem. This ferritin nanoparticle vaccine technology represents a novel, safe and immunogenic platform with potential application for pandemic preparedness and universal influenza vaccine development.

Recombinant respiratory syncytial virus F protein expression is hindered by inefficient nuclear export and mRNA processing.

Studies of the fusion activity of respiratory syncytial virus (RSV) F protein are significantly hindered by low recombinant expression levels. While infection produces F protein levels detectable by western blot, recombinant expression produces undetectable to low levels of F protein. Identifying the obstacles that hinder recombinant F protein expression may lead to improved expression and facilitate the study of F protein function. We hypothesized that nuclear localization and/or inefficient RNA polymerase II-mediated transcription contribute to poor recombinant F protein expression. This study shows a combination of stalled nuclear export, premature polyadenylation, and low mRNA abundance all contribute to low recombinant F protein expression levels. In addition, this study provides an expression optimization strategy that results in greater F protein expression levels than observed by codon-optimization of the F protein gene, which will be useful for future studies of F protein function.

Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA.

BACKGROUND: Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks. RESULTS: A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus. CONCLUSION: These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.

Palivizumab epitope-displaying virus-like particles protect rodents from RSV challenge.

Respiratory syncytial virus (RSV) is the most common cause of serious viral bronchiolitis in infants, young children, and the elderly. Currently, there is not an FDA-approved vaccine available for RSV, though the mAb palivizumab is licensed to reduce the incidence of RSV disease in premature or at-risk infants. The palivizumab epitope is a well-characterized, approximately 24-aa helix-loop-helix structure on the RSV fusion (F) protein (F254-277). Here, we genetically inserted this epitope and multiple site variants of this epitope within a versatile woodchuck hepadnavirus core-based virus-like particle (WHcAg-VLP) to generate hybrid VLPs that each bears 240 copies of the RSV epitope in a highly immunogenic arrayed format. A challenge of such an epitope-focused approach is that to be effective, the conformational F254-277 epitope must elicit antibodies that recognize the intact virus. A number of hybrid VLPs containing RSV F254-277 were recognized by palivizumab in vitro and elicited high-titer and protective neutralizing antibody in rodents. Together, the results from this proof-of-principle study suggest that the WHcAg-VLP technology may be an applicable approach to eliciting a response to other structural epitopes.

A host-range restricted parainfluenza virus type 3 (PIV3) expressing the human metapneumovirus (hMPV) fusion protein elicits protective immunity in African green monkeys.

Human metapneumovirus (hMPV) infection causes respiratory tract disease similar to that observed during human respiratory syncytial virus infection (hRSV). hMPV infections have been reported across the entire age spectrum although the most severe disease occurs in young children. No vaccines, chemotherapeutics or antibodies are presently available for preventing or treating hMPV infections. In this study, a bovine/human chimeric parainfluenza virus type 3 (b/h PIV3) expressing the human parainfluenza type 3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins was engineered to express hMPV fusion (F) protein from the second genome position (b/h PIV3/hMPV F2) with the goal of generating a novel hMPV vaccine. b/h PIV3/hMPV F2 was previously shown to protect hamsters from challenge with wt hMPV (Tang RS, Schickli JH, Macphail M, Fernandes F, Bicha L, Spaete J, et al. Effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfluenza virus type 3 on virus replication and immunogenicity. J Virol 2003;77:10819-28) and is here further evaluated for efficacy and immunogenicity in African green monkeys (AGMs). AGMs immunized intranasally and intratracheally with b/h PIV3/hMPV F2 generated hMPV- and hPIV3-specific humoral and cellular immune responses and were protected from wt hMPV infection. In a separate study, the host-range restriction of b/h PIV3/hMPV F2 replication relative to wt hPIV3 was performed in rhesus monkeys to demonstrate attenuation. These studies showed that b/h PIV3/hMPV F2 was immunogenic, protective and attenuated in non-human primates and warrants further evaluation in humans as a vaccine candidate for prevention of hMPV-associated respiratory tract diseases.

Parainfluenza virus type 3 expressing the native or soluble fusion (F) Protein of Respiratory Syncytial Virus (RSV) confers protection from RSV infection in African green monkeys.

Respiratory syncytial virus (RSV) causes respiratory disease in young children, the elderly, and immunocompromised individuals, often resulting in hospitalization and/or death. After more than 40 years of research, a Food and Drug Administration-approved vaccine for RSV is still not available. In this study, a chimeric bovine/human (b/h) parainfluenza virus type 3 (PIV3) expressing the human PIV3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins from an otherwise bovine PIV3 (bPIV3) genome was employed as a vector for RSV antigen expression with the aim of generating novel RSV vaccines. b/h PIV3 vaccine candidates expressing native or soluble RSV F proteins were evaluated for efficacy and immunogenicity in a nonhuman primate model. b/h PIV3 is suited for development of pediatric vaccines since bPIV3 had already been evaluated in clinical studies in 1- and 2-month-old infants and was found to be safe, immunogenic, and nontransmissible in a day care setting (Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Lee et al., J. Infect. Dis. 184:909-913, 2001). African green monkeys immunized with b/h PIV3 expressing either the native or soluble RSV F protein were protected from challenge with wild-type RSV and produced RSV neutralizing and RSV F-protein specific immunoglobulin G serum antibodies. The PIV3-vectored RSV vaccines evaluated here further underscore the utility of this vector system for developing safe and immunogenic pediatric respiratory virus vaccines.