The local tumour immune response following systemic Salmonella enterica serovar Typhimurium infection

In recent years, there is renewed interest in the potential of bacteria as an alternative cancer therapeutic strategy. Salmonella enterica serovar Typhimurium is arguably the most well studied strain of bacteria for cancer therapy, examined in both pre-clinical and clinical settings. Many of the studies which have demonstrated a role for S. Typhimurium in tumour growth inhibition or regression have focused on increasing the tumour-specific localisation of the bacteria, or enhancing the efficacy of the treatment modality. However, the exact mechanisms underlying S. Typhimurium-mediated tumour growth inhibition are incompletely elucidated, particularly with respect to the myeloidderived immune cells, such as monocytes and macrophages. The current study intended to address the dearth of information in the literature pertaining to the overall tumour-local immune response to systemically administered S. Typhimurium. This was achieved through the development of an in vivo tumour model and the optimisation of the S. Typhimurium administration protocol to maximise therapeutic effect. This allowed for the investigation of changes in multiple immune cell types in the tumour, both in number and functional phenotype, following infection. It was found that following systemic SL7207 infection, there was an increase in the secretion of pro-inflammatory mediators in the tumour milieu. This was accompanied by the activation of both neutrophils and monocytes, and possibly increased migration of tumour-associated dendritic cells. Interestingly, we found evidence to suggest that resident tumour-associated macrophages (TAMs) do not participate in mediating the pro-inflammatory tumour microenvironment following infection, which is suggested in some published reports. We were also interested in the types of T cell responses stimulated in the tumour following infection. This investigation revealed increases in the frequency of tumour-associated T helper (TH)1, but also TH17 cells following infection. There was also a concomitant decrease in the frequency of the tumour-promoting, T regulatory (Treg), cells in the tumour mass. To our knowledge, this is the first report to suggest a role for either TH17 or Tregs in playing a role in bacterial-mediated cancer therapy. Given the phenotypic changes in the tumour-associated monocytes following infection, we chose to assess the contribution of this cell population to S. Typhimurium-mediated tumour growth inhibition. This was attempted though the employment of transgenic mice lacking circulating monocytes and clodronate liposome-mediated depletion of monocytes/macrophages. Both of these approaches were proficient in depleting tumour monocytes in the uninfected state, with TAMs also affected by clodronate liposome treatment. However, we found that neither of these approaches was sufficient to mediate the depletion of tumour-recruited monocytes following systemic S. Typhimurium infection. Interestingly, clodronate liposome treatment abrogated the S. Typhimurium-induced tumour growth inhibitory effects anyway. Upon further investigation, it was observed that the spleens of clodronate liposome-treated mice that were systemically infected with S. Typhimurium did not experience splenomegaly like their control PBS liposome-treated counterparts. As the spleen is a source of systemic inflammatory mediators following infection and splenic monocytes contribute to the tumour monocyte/macrophage population, the current hypothesis is that the splenic monocytes mediate tumour-growth inhibition in S. Typhimurium infected mice. This concept antagonises the prevailing ideology in the literature that tumour-local immune cells are the effectors of bacterial-mediated tumour growth arrest. This study also sought to enhance the tumour arrest effects of S. Typhimurium through transformation of the bacteria with a eukaryotic expression vector encoding tumour inhibitory genes, destined for transfer to the tumour cells. However, through this investigation, it was discovered that the bacteria transformed with such a plasmid exhibited an aberrant morphology and phenotype, which we subsequently discovered was due to a phage origin of replication encoded in the plasmid. The data generated in this thesis provides valuable information pertaining to the general immune response in the tumour following systemically administered S. Typhimurium. Furthermore, we propose a role for monocytes, possibly of splenic origin, in mediating the effects of S. Typhimurium-induced tumour growth inhibition. Finally, we identified a feature of eukaryotic expression plasmid, a phage origin of replication, which is not compatible with S. Typhimurium and should be avoided for bactofection, and other bacteriological, studies

The antibody response to SARS-CoV-2 Beta underscores the antigenic distance to other variants

Alpha-B.1.1.7, Beta-B.1.351, Gamma-P.1 and Delta-B.1.617.2 variants of SARS-CoV-2 express multiple mutations in the spike protein (S). These may alter the antigenic structure of S, causing escape from natural or vaccine-induced immunity. Beta is particularly difficult to neutralize using serum induced by early pandemic SARS-CoV-2 strains and is most antigenically separated from Delta. To understand this, we generated 674 mAbs from Beta infected individuals and performed a detailed structure-function analysis of the 27 most potent mAbs: one binding the spike N-terminal domain (NTD), the rest the receptor binding domain (RBD). Two of these RBD-binding mAbs recognise a neutralizing epitope conserved between SARS-CoV-1 and -2, whilst 18 target mutated residues in Beta: K417N, E484K, and N501Y. There is a major response to N501Y including a public IgVH4-39 sequence, with E484K and K417N also targeted. Recognition of these key residues underscores why serum from Beta cases poorly neutralizes early pandemic and Delta viruses

Potent cross-reactive antibodies following Omicron breakthrough in vaccinees

Highly transmissible Omicron variants of SARS-CoV-2 currently dominate globally. Here, we compare neutralization of Omicron BA.1, BA.1.1 and BA.2. BA.2 RBD has slightly higher ACE2 affinity than BA.1 and slightly reduced neutralization by vaccine serum, possibly associated with its increased transmissibility. Neutralization differences between sub-lineages for mAbs (including therapeutics) mostly arise from variation in residues bordering the ACE2 binding site, however, more distant mutations S371F (BA.2) and R346K (BA.1.1) markedly reduce neutralization by therapeutic antibody Vir-S309. In-depth structure-and-function analyses of 27 potent RBD-binding mAbs isolated from vaccinated volunteers following breakthrough Omicron-BA.1 infection reveals that they are focussed in two main clusters within the RBD, with potent right-shoulder antibodies showing increased prevalence. Selection and somatic maturation have optimized antibody potency in less-mutated epitopes and recovered potency in highly mutated epitopes. All 27 mAbs potently neutralize early pandemic strains and many show broad reactivity with variants of concern

A delicate balance between antibody evasion and ACE2 affinity for Omicron BA.2.75

Variants of SARS CoV-2 have caused successive global waves of infection. These variants, with multiple mutations in the spike protein are thought to facilitate escape from natural and vaccine-induced immunity and often increase in the affinity for ACE2. The latest variant to cause concern is BA.2.75, identified in India where it is now the dominant strain, with evidence of wider dissemination. BA.2.75 is derived from BA.2 and contains four additional mutations in the receptor binding domain (RBD). Here we perform an antigenic and biophysical characterization of BA.2.75, revealing an interesting balance between humoral evasion and ACE2 receptor affinity. ACE2 affinity for BA.2.75 is increased 9-fold compared to BA.2; there is also evidence of escape of BA.2.75 from immune serum, particularly that induced by Delta infection which may explain the rapid spread in India, where BA.2.75 is now the dominant variant. ACE2 affinity appears to be prioritised over greater escape

Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum

The Omicron lineage of SARS-CoV-2, first described in November 2021, spread rapidly to become globally dominant and has split into a number of sub-lineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africa’s Gauteng region uncovered two new sub-lineages, BA.4 and BA.5 which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences and, although closely related to BA.2, contain further mutations in the receptor binding domain of spike. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by serum from triple AstraZeneca or Pfizer vaccinated individuals compared to BA.1 and BA.2. Furthermore, using serum from BA.1 vaccine breakthrough infections there are likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations

Two doses of SARS-CoV-2 vaccination induce robust immune responses to emerging SARS-CoV-2 variants of concern

The extent to which immune responses to natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immunization with vaccines protect against variants of concern (VOC) is of increasing importance. Accordingly, here we analyse antibodies and T cells of a recently vaccinated, UK cohort, alongside those recovering from natural infection in early 2020. We show that neutralization of the VOC compared to a reference isolate of the original circulating lineage, B, is reduced: more profoundly against B.1.351 than for B.1.1.7, and in responses to infection or a single dose of vaccine than to a second dose of vaccine. Importantly, high magnitude T cell responses are generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. Vaccination is required to generate high potency immune responses to protect against these and other emergent variants

Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum

SARS-CoV-2 has undergone progressive change with variants conferring advantage rapidly becoming dominant lineages e.g. B.1.617. With apparent increased transmissibility variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the UK. Here we study the ability of monoclonal antibodies, convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2 and complement this with structural analyses of Fab/RBD complexes and map the antigenic space of current variants. Neutralization of both viruses is reduced when compared with ancestral Wuhan related strains but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2 suggesting that individuals previously infected by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insight for immunisation policy with future variant vaccines in non-immune populations