Development and operation of the defence COVID-19 lab as a SARS-CoV-2 diagnostic screening capability for UK military personnel.

BACKGROUND: In the face of the COVID-19 pandemic, the Defence Science and Technology Laboratory (Dstl) and Defence Pathology combined to form the Defence Clinical Lab (DCL), an accredited (ISO/IEC 17025:2017) high-throughput SARS-CoV-2 PCR screening capability for military personnel. LABORATORY STRUCTURE AND RESOURCE: The DCL was modular in organisation, with laboratory modules and supporting functions combining to provide the accredited SARS-CoV-2 (envelope (E)-gene) PCR assay. The DCL was resourced by Dstl scientists and military clinicians and biomedical scientists. LABORATORY RESULTS: Over 12 months of operation, the DCL was open on 289 days and tested over 72 000 samples. Six hundred military SARS-CoV-2-positive results were reported with a median E-gene quantitation cycle (Cq) value of 30.44. The lowest Cq value for a positive result observed was 11.20. Only 64 samples (0.09%) were voided due to assay inhibition after processing started. CONCLUSIONS: Through a sustained effort and despite various operational issues, the collaboration between Dstl scientific expertise and Defence Pathology clinical expertise provided the UK military with an accredited high-throughput SARS-CoV-2 PCR test capability at the height of the COVID-19 pandemic. The DCL helped facilitate military training and operational deployments contributing to the maintenance of UK military capability. In offering a bespoke capability, including features such as testing samples in unit batches and oversight by military consultant microbiologists, the DCL provided additional benefits to the UK Ministry of Defence that were potentially not available from other SARS-CoV-2 PCR laboratories. The links between Dstl and Defence Pathology have also been strengthened, benefitting future research activities and operational responses.

Comparative Efficacy of Intramuscular and Scarification Routes of Administration of Live Smallpox Vaccine in a Murine Challenge Model.

In recent years concern has mounted regarding the possibility of a re-emergence of smallpox through biowarfare or bioterrorism. There is also concern over the incidence of human monkeypox in endemic areas and the potential for monkeypox to be accidentally transported to non-endemic areas. In the event of re-emergence of smallpox or emergence of monkeypox, the accepted route of administration for live replicating smallpox vaccine is dermal scarification, which generates a virus-shedding lesion that persists for several days at the vaccination site. The lesion is a potential source of contact transmission of vaccine to individuals who may be contra-indicated for receipt of the live vaccine. In this study, we compare dermal scarification with intramuscular vaccination for replicating smallpox vaccine in a mouse lethal challenge model. Comparisons are made over multiple vaccine and challenge doses and data recorded for lethality, disease severity, and antibody responses. Qualitative and quantitative differences between the two routes are observed, and for the intramuscular route the febrile response is not suppressed after subsequent virulent vaccinia virus challenge. However both routes generate an immune response and protect from severe disease and death. Although dermal scarification is the preferred route of vaccination for the general population, intramuscular vaccination may be an option for people who are not contraindicated for the live vaccine, but who are close contacts of people who are contraindicated for the live vaccine, in an emergency situation.

Isolation of single-chain antibody fragments against Venezuelan equine encephalomyelitis virus from two different immune sources.

Venezuelan equine encephalomyelitis (VEE) virus is an important human and veterinary pathogen of Central and South America. The virus can cause widespread epidemics, affecting hundreds of thousands of horses, and thousands of humans. Detection of the virus early in infection and in mosquito populations may allow epidemics to be predicted such that suitable prophylaxis, such as vaccination, can be used to reduce disease severity and transmission. The sensitivity and specificity of current immunoassays, based on conventional monoclonal and polyclonal antibodies, needs to be improved for the diagnosis of infection. We have examined phage display libraries expressing single-chain antibodies (scFv) produced from two different immune sources, a hybridoma cell line and an immunized mouse spleen. The libraries were panned against VEE virus to select for specific scFvs. scFvs isolated from both libraries were specific for the same epitope on the VEE virus and sequence analysis showed that the scFvs were almost identical apart from the CDR3 region of the heavy chain. The data presented in this article suggest that although scFvs may be useful tools for the detection of viruses, there are serious limitations with the use of phage display as a tool for the isolation of specific antibodies.

A carpet-based mechanism for direct antimicrobial peptide activity against vaccinia virus membranes.

Antimicrobial peptides have activity against a wide variety of biological membranes and are an important component of innate immunity in vertebrate as well as invertebrate systems. The mechanisms of action of these peptides are incompletely understood and a number of competing but not necessarily mutually exclusive models exist. In this study we examined the virucidal activity of four peptides, the human cathelicidin derived LL37, Xenopus alanine-substituted Magainin-2 amide, uperin-3.1, and a cecropin-LL37 hybrid against vaccinia virus. The peptides were shown to be differentially virucidal but all were shown to attack the viral envelope, with LL37 being the most effective and uperin-3.1 the least. Density gradient analysis of the treated virions indicated the virus outer membrane was efficiently removed by peptide action and suggests a mechanism of direct virus inactivation that is consistent with the carpet model for peptide-mediated membrane disruption. Interestingly, the least effective peptide uperin-3.1 was equally effective as the others at inducing susceptibility to neutralizing antibody. This suggests that in addition to direct killing by a carpet-based mechanism, the peptides may simultaneously operate a different mechanism that exposes sequestered antigen without membrane removal.

Comparative efficacy of modified vaccinia Ankara (MVA) as a potential replacement smallpox vaccine.

International concern over the potential consequences of a Bioterrorist or Biowarfare associated release of variola virus have prompted renewed interest in the vaccines for smallpox. The traditional live, replicating vaccine strains are subject to novel safety concerns associated with historical production methods in domesticated ruminants and the additional hazards that vaccinia virus poses for people with immune system abnormalities or a history of eczematous skin conditions. In this study we have examined the longevity and efficacy of immunity induced by a non-replicating smallpox vaccine candidate, modified vaccinia Ankara (MVA) in a murine model using intranasal and aerosol routes of infection. Two-step vaccinations of MVA followed by traditional Lister vaccine are compared with either Lister alone or MVA alone, and the longevity of the protection induced by MVA is assessed. MVA is found to be broadly similar to Lister. Although protection is shown to decay with time, when administered at a standard human dose the longevity of protection induced by MVA is comparable to that induced by Lister.

Comparative efficacy of replicating smallpox vaccine strains in a murine challenge model.

There is currently considerable concern about the vulnerability of human populations to biowarfare or bioterrorist attacks with variola virus (VARV). Traditional smallpox vaccines were manufactured using the lymph of ruminants infected with the vaccinia virus (VACV). However, these production methods do not meet current standards for vaccines, especially since the emergence of transmissable spongiform encephalopathies in domesticated ruminants. This study has examined the protective efficacy of the Lister (Elstree) vaccine strain from various sources in a murine lethal challenge model. Considerable variation in efficacy is observed between the Lister material obtained from the American Type Culture Collection (ATCC) and the same strain obtained from vaccine stockpiles. A new, tissue-culture derived Lister vaccine is assessed against a bench-mark of multiple lots from a historical stockpile of the traditional vaccine. Apparent qualitative differences are observed between historical and new vaccines. Statistically significant differences are observed between different batches of the traditional vaccine, and the efficacy of the tissue-culture produced vaccine falls within this range.

Palmitylation of the vaccinia virus 37-kDa major envelope antigen. Identification of a conserved acceptor motif and biological relevance.

Computer-assisted alignment of known palmitylproteins was used to identify a potential peptide motif, TMDX1-12AAC(C)A (TMD, transmembrane domain; X, any amino acid; C, cysteine acceptor residues; A, aliphatic residue) responsible for directing internal palmitylation of the vaccinia virus 37-kDa major envelope antigen, p37. Site-directed mutagenesis was used to confirm this motif as the site of modification and to produce a nonpalmitylated version of the p37 protein. Comparative phenotypic analysis of the wild-type and mutant p37 alleles confirmed that the p37 protein is involved in viral envelopment and egress, and suggested that attachment of the palmitate moiety was essential for correct intracellular targeting and protein function.

Identification of two T-cell epitopes on the candidate Epstein-Barr virus vaccine glycoprotein gp340 recognized by CD4+ T-cell clones.

Current efforts to develop an Epstein-Barr virus subunit vaccine are based on the major envelope glycoprotein gp340. Given the central role of CD4+ T cells in regulating immune responses to subunit vaccine antigens, the present study has begun the work of identifying linear epitopes which are recognized by human CD4+ T cells within the 907-amino-acid sequence of gp340. A panel of gp340-specific CD4+ T-cell clones from an Epstein-Barr virus-immune donor were first assayed for their proliferative responses to a series of truncated gp340 molecules expressed from recombinant DNA vectors in rat GH3 cells, by using an autologous B lymphoblastoid cell line as a source of antigen-presenting cells. The first four T-cell clones analyzed all responded to a truncated form of gp340 which contained only the first 260 N-terminal amino acids. These clones were subsequently screened for responses to each of a panel of overlapping synthetic peptides (15-mers) corresponding to the primary amino acid sequence of the first 260 N-terminal amino acids of gp340. One clone (CG2.7) responded specifically to peptides from the region spanning amino acids 61 to 81, while three other clones (CG5.15, CG5.24, and CG5.36) responded specifically to peptides from the region spanning amino acids 163 to 183. Work with individual peptides from these regions allowed finer mapping of the T-cell epitopes and also revealed the highly dose-dependent nature of peptide-induced responses, with inhibitory effects apparent when the most antigenic peptides were present at supraoptimal concentrations. Experiments using homozygous typing B lymphoblastoid cell lines as antigen-presenting cells showed that the T-cell clones with different epitope specificities were restricted through different HLA class II antigens; clone CG2.7 recognized epitope 61-81 in the context of HLA DRw15, whereas clones CG5.15, CG5.24, and CG5.36 recognized epitope 163-183 in the context of HLA DRw11. The present protocol therefore makes a systematic analysis of CD4+ T-cell epitopes within gp340 possible; it will be necessary to screen gp340-specific T-cell clones from a variety of donors to assess the wider influence of HLA class II polymorphism upon epitope choice.

Immunogenic and antigenic epitopes of immunoglobulins. XIV. Antigenic variants of IgG4 proteins revealed with monoclonal antibodies.

Two IgG4 paraproteins having heavy chains of normal molecular weight are shown to be antigenically distinct in their reactivity profiles with 18 monoclonal antibodies having specificity for the IgG2 or IgG4 subclasses. One protein expresses IgG2 and IgG4 epitopes within the C gamma 2 domain. A second protein is deficient in the expression of IgG4 Fc-specific epitopes. These proteins will be of value in defining the structural basis of Fc effector functions and individual epitope expression.