kenneth Walker - Academia.edu (original) (raw)

Papers by kenneth Walker

Journal of General Virology, 1998

Vaccine, 2006

The lack of unequivocal immunological correlates of human protection and an absence of a validate... more The lack of unequivocal immunological correlates of human protection and an absence of a validated animal model for acellular pertussis vaccines, compounded by limited opportunity to undertake efficacy studies in humans and laboratory evaluation side by side, has made it difficult to compare vaccines and formulations. In the present study, the effect on the booster response to pertussis in adolescents primed in infancy with whole cell pertussis vaccine, of three low dose acellular pertussis/diphtheria/tetanus toxoid (TdPa) formulations with or without inactivated poliomyelitis vaccine (IPV) components, was investigated. To assess the relationship between laboratory vaccine evaluation and clinical trial performance, parallel evaluation of the same TdPa vaccines were carried out in a mouse booster model with whole cell pertussis vaccine priming. Prior to boosting, the clinical subjects had low cell mediated immune responses (CMI) responses to pertussis vaccine components. After boosting, all TdPa formulations stimulated CMI responses to the pertussis vaccine components assessed. The booster responses to the pertussis antigens remained skewed towards Th1 type even though acellular pertussis vaccines were used. In general the antibody and CMI responses to pertussis antigens in the mouse model followed the trend seen in the human subjects. Protection against aerosol challenge with virulent Bordetella pertussis was related to the magnitude of the antibody and CMI responses in the mouse model. As in the human subjects, the responses remained skewed towards Th1 type.

Infection and Immunity, 2005

Intravenous immunoglobulin (IVIg) is used to treat patients with primary antibody deficiencies an... more Intravenous immunoglobulin (IVIg) is used to treat patients with primary antibody deficiencies and, at high doses, to treat a range of autoimmune and inflammatory disorders. With high-dose IVIg (hdIVIg), immunomodulatory mechanisms act on a range of cells, including T cells, B cells, and dendritic cells. Here, we demonstrate that the treatment of M. tuberculosis -infected mice with a single cycle of hdIVIg resulted in substantially reduced bacterial loads in the spleen and lungs when administered at either an early or late stage of infection. Titration of the IVIg showed a clear dose-response effect. There was no reduction in bacterial load when mice were given equimolar doses of another human protein, human serum albumin, or maltose, the stabilizing agent in the IVIg preparation. HdIVIg in vitro had no inhibitory effect on the growth of M. tuberculosis in murine bone marrow-derived macrophages. In addition, the effect of hdIVIg on bacterial loads was not observed in nude mice, sugg...

Biochemical Society Transactions, 1997

Comparison of peripheral blood phenotype in pathogenic and attenuated SIV infection.

Methods in Molecular Biology, 2009

Antibody titre is a measure of the presence and amount of antibodies specific to an antigen that ... more Antibody titre is a measure of the presence and amount of antibodies specific to an antigen that are present in the blood. In particular, the titre of an antibody sample is a measure of the antibody concentration determined under a defined set of conditions, with the antibody concentration being commonly established by enzyme-linked immunosorbent assay, also called ELISA, or a variation of this technology.Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. The ELISA/EIA approach is an extremely robust technology and readily amenable to validation and quality control if adherence to ISO quality standards is required. This chapter describes in detail a specific ELISA protocol which has potential generic application.

Bioprocess, Bioseparation, and Cell Technology, 2009

As depicted in Fig. 1, cells of the monocytic lineage arise from pluripotent stem cells in adult ... more As depicted in Fig. 1, cells of the monocytic lineage arise from pluripotent stem cells in adult bone marrow and are released into the blood, where they are observed pre-dominantly as cells of monocytic morphology. Monocytes constitute approximately 5–10% of peripheral blood ...

Bioprocess, Bioseparation, and Cell Technology, 2009

The immune system can be divided into two major arms of immune activity based on functional crite... more The immune system can be divided into two major arms of immune activity based on functional criteria; the immune functions being (i) cell-mediated immunity and (ii) humoral immunity. The cells mediating these activities are distinct and specific to the type of immune response ...

Journal of Immunological Methods, 1998

Many normal and pathological processes have been shown to be influenced by the quantitative and q... more Many normal and pathological processes have been shown to be influenced by the quantitative and qualitative nature of cytokine production. Cytokines have also been shown to be central in modulating host responses to invasion by pathogens. Therefore detection and analysis of cytokine production are critical in the understanding of host responses to a variety of immunological insults. The detection of cytokines by bioassay or immunoassay provides data from in vitro experiments and can be used to determine the circulating plasma levels of particular cytokines. However, many investigations require the detection of cytokines in small numbers of cells or directly from tissue samples. It is in this particular area that detection and analysis of cytokine gene expression by use of the sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) has application. The purpose of these protocols is to provide a practical guide to the detection of cytokine gene expression using RT-PCR techniques. Procedures for analysing mRNA expression in cell culture. cell suspensions and tissue samples will be described and pitfalls and caveats concerning the techniques will be discussed. These protocols should provide a starting point for the development of RT-PCR technology in laboratories with only limited molecular experience.

Journal of Biological Chemistry, 2011

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tubercu... more One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis.

Immunology Letters, 2005

Expression of the normal form of prion protein (PrP C) has been reported on a wide range cells in... more Expression of the normal form of prion protein (PrP C) has been reported on a wide range cells including lymphocytes and antigen presenting cells, however the functional role of PrP C remains to be fully elucidated. Here we report the effect of reintroducing the PrP gene into splenocytes derived from prion knockout (PrP 0/0) mice and comparing their responses with splenocytes lacking a functional PrP gene. Reintroduction of the PrP gene was carried out by transfecting cells with pC1PrPEH, a plasmid expressing mouse PrP. Following transfection, T cells demonstrated an increased capacity to proliferate in response to ConA and PMA/ionomycin compared to T cells lacking the functional PrP gene. A bioassay used to determine IL-2 levels indicated that the reintroduction of the PrP gene might enhance IL-2 expression in response to ConA. Levels of IFN-␥ produced also showed an increase following transfection with PrP expressing plasmid. A comparison between splenocytes derived from PrP 0/0 and PrP +/+ also demonstrated some differences in cytokine production and proliferation. Together these results show PrP C has an impact on the normal T cell activation and proliferation in response to mitogens and also potentially antigen responsiveness.

Current Molecular Medicine, 2007

Heat shock proteins (hsps) are a highly conserved family of proteins, first recognized by their u... more Heat shock proteins (hsps) are a highly conserved family of proteins, first recognized by their upregulated expression in response to host exposure to raised temperatures. Further study has revealed that they have numerous functions in the cell, primarily as chaperones mediating both the correct folding of nascent polypeptide chains and the dissolution of aggregated protein complexes. The energy requirement for this chaperone activity is provided by the ATPase activity found in most families of hsps and thus the peptide binding capacity is controlled by ATP hydrolysis. The structural consequence of this is that hsps isolated in situ are found complexed to chaperoned peptides (hspCs). Much previous work has implicated hsps in the immune response to pathogens and recent studies have shown that the interaction of hsps with antigen presenting cells, such as dendritic cells (DCs), mediates the integration of the innate and acquired immune responses. This central role for hspCs in immunity is facilitated by their dual function in both innate immunity, with the induction of cytokines and the maturation of DCs mediated by the hsp component, and acquired immunity, with the trafficking of antigens chaperoned in hspCs for antigen presentation by the mature DCs.

Biochemical Society Transactions, 2004

The need for an effective TB (tuberculosis) vaccine remains acute, with tuberculosis still one of... more The need for an effective TB (tuberculosis) vaccine remains acute, with tuberculosis still one of the major killers worldwide and 3 million new infections annually. We report here on the immune responses elicited by HspCs (heat-shock protein–peptide complexes) isolated from BCG (Bacille Calmette–Guérin) vaccine. These HspCs elicit both the appropriate cellular and protective immune responses required to merit their further development as TB vaccine candidates.

The Journal of Infectious Diseases, 2012

Clinical and Experimental Immunology, 2004

SUMMARY The in -vivo clearance of Bordetella pertussis infections in murine models in naive mice ... more SUMMARY The in -vivo clearance of Bordetella pertussis infections in murine models in naive mice and animals vaccinated with whole-cell vaccine is considered to be via a Th-1-dependent mechanism in which interleukin-12 (IL)-12 may play a prominent role. It has also been demonstrated clearly that the treatment of animals with macrophage-derived IL-12 administered with an acellular vaccine can increase the efficacy of this vaccine preparation to levels seen with the whole-cell vaccine. However, the effects of exogenously added IL-12 on immune responses in non-vaccinated B. pertussis-challenged mice remain unclear, with two studies giving contradictory findings. In this study we have treated mice with escalating doses of mIL-12 (0·1–10 µg/mouse) prior to challenge with B. pertussis (using an aerosol challenge model of infection). The ability of mice to clear infection was assessed in IL-12 treated and in phosphate buffered saline (PBS) control animals at days 6 and 13 post-challenge. L...

BMC infectious diseases, Aug 12, 2016

In the absence of a validated animal model and/or an immune correlate which predict vaccine-media... more In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed. We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen. Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57...

This article cites 32 articles, 12 of which can be accessed free

Tuberculosis, 2016

The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brough... more The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brought together a wide and diverse community involved in tuberculosis vaccine research and development to discuss the current status of, and future directions for this critical effort. This paper summarizes the sessions on Low-Dose NHP Challenge Models, Novel Approaches to Animal Models for TB Vaccine R&D, Novel Antigen Delivery Strategies, and Next Generation TB Vaccines and Vaccine Concepts. Summaries of all sessions from the 4th Global Forum are compiled in a special supplement of Tuberculosis. [July 2016, Vol 99, Supp S1 - XX].

Journal of General Virology, 1998

Vaccine, 2006

The lack of unequivocal immunological correlates of human protection and an absence of a validate... more The lack of unequivocal immunological correlates of human protection and an absence of a validated animal model for acellular pertussis vaccines, compounded by limited opportunity to undertake efficacy studies in humans and laboratory evaluation side by side, has made it difficult to compare vaccines and formulations. In the present study, the effect on the booster response to pertussis in adolescents primed in infancy with whole cell pertussis vaccine, of three low dose acellular pertussis/diphtheria/tetanus toxoid (TdPa) formulations with or without inactivated poliomyelitis vaccine (IPV) components, was investigated. To assess the relationship between laboratory vaccine evaluation and clinical trial performance, parallel evaluation of the same TdPa vaccines were carried out in a mouse booster model with whole cell pertussis vaccine priming. Prior to boosting, the clinical subjects had low cell mediated immune responses (CMI) responses to pertussis vaccine components. After boosting, all TdPa formulations stimulated CMI responses to the pertussis vaccine components assessed. The booster responses to the pertussis antigens remained skewed towards Th1 type even though acellular pertussis vaccines were used. In general the antibody and CMI responses to pertussis antigens in the mouse model followed the trend seen in the human subjects. Protection against aerosol challenge with virulent Bordetella pertussis was related to the magnitude of the antibody and CMI responses in the mouse model. As in the human subjects, the responses remained skewed towards Th1 type.

Infection and Immunity, 2005

Intravenous immunoglobulin (IVIg) is used to treat patients with primary antibody deficiencies an... more Intravenous immunoglobulin (IVIg) is used to treat patients with primary antibody deficiencies and, at high doses, to treat a range of autoimmune and inflammatory disorders. With high-dose IVIg (hdIVIg), immunomodulatory mechanisms act on a range of cells, including T cells, B cells, and dendritic cells. Here, we demonstrate that the treatment of M. tuberculosis -infected mice with a single cycle of hdIVIg resulted in substantially reduced bacterial loads in the spleen and lungs when administered at either an early or late stage of infection. Titration of the IVIg showed a clear dose-response effect. There was no reduction in bacterial load when mice were given equimolar doses of another human protein, human serum albumin, or maltose, the stabilizing agent in the IVIg preparation. HdIVIg in vitro had no inhibitory effect on the growth of M. tuberculosis in murine bone marrow-derived macrophages. In addition, the effect of hdIVIg on bacterial loads was not observed in nude mice, sugg...

Biochemical Society Transactions, 1997

Comparison of peripheral blood phenotype in pathogenic and attenuated SIV infection.

Methods in Molecular Biology, 2009

Antibody titre is a measure of the presence and amount of antibodies specific to an antigen that ... more Antibody titre is a measure of the presence and amount of antibodies specific to an antigen that are present in the blood. In particular, the titre of an antibody sample is a measure of the antibody concentration determined under a defined set of conditions, with the antibody concentration being commonly established by enzyme-linked immunosorbent assay, also called ELISA, or a variation of this technology.Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. The ELISA/EIA approach is an extremely robust technology and readily amenable to validation and quality control if adherence to ISO quality standards is required. This chapter describes in detail a specific ELISA protocol which has potential generic application.

Bioprocess, Bioseparation, and Cell Technology, 2009

As depicted in Fig. 1, cells of the monocytic lineage arise from pluripotent stem cells in adult ... more As depicted in Fig. 1, cells of the monocytic lineage arise from pluripotent stem cells in adult bone marrow and are released into the blood, where they are observed pre-dominantly as cells of monocytic morphology. Monocytes constitute approximately 5–10% of peripheral blood ...

Bioprocess, Bioseparation, and Cell Technology, 2009

The immune system can be divided into two major arms of immune activity based on functional crite... more The immune system can be divided into two major arms of immune activity based on functional criteria; the immune functions being (i) cell-mediated immunity and (ii) humoral immunity. The cells mediating these activities are distinct and specific to the type of immune response ...

Journal of Immunological Methods, 1998

Many normal and pathological processes have been shown to be influenced by the quantitative and q... more Many normal and pathological processes have been shown to be influenced by the quantitative and qualitative nature of cytokine production. Cytokines have also been shown to be central in modulating host responses to invasion by pathogens. Therefore detection and analysis of cytokine production are critical in the understanding of host responses to a variety of immunological insults. The detection of cytokines by bioassay or immunoassay provides data from in vitro experiments and can be used to determine the circulating plasma levels of particular cytokines. However, many investigations require the detection of cytokines in small numbers of cells or directly from tissue samples. It is in this particular area that detection and analysis of cytokine gene expression by use of the sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) has application. The purpose of these protocols is to provide a practical guide to the detection of cytokine gene expression using RT-PCR techniques. Procedures for analysing mRNA expression in cell culture. cell suspensions and tissue samples will be described and pitfalls and caveats concerning the techniques will be discussed. These protocols should provide a starting point for the development of RT-PCR technology in laboratories with only limited molecular experience.

Journal of Biological Chemistry, 2011

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tubercu... more One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis.

Immunology Letters, 2005

Expression of the normal form of prion protein (PrP C) has been reported on a wide range cells in... more Expression of the normal form of prion protein (PrP C) has been reported on a wide range cells including lymphocytes and antigen presenting cells, however the functional role of PrP C remains to be fully elucidated. Here we report the effect of reintroducing the PrP gene into splenocytes derived from prion knockout (PrP 0/0) mice and comparing their responses with splenocytes lacking a functional PrP gene. Reintroduction of the PrP gene was carried out by transfecting cells with pC1PrPEH, a plasmid expressing mouse PrP. Following transfection, T cells demonstrated an increased capacity to proliferate in response to ConA and PMA/ionomycin compared to T cells lacking the functional PrP gene. A bioassay used to determine IL-2 levels indicated that the reintroduction of the PrP gene might enhance IL-2 expression in response to ConA. Levels of IFN-␥ produced also showed an increase following transfection with PrP expressing plasmid. A comparison between splenocytes derived from PrP 0/0 and PrP +/+ also demonstrated some differences in cytokine production and proliferation. Together these results show PrP C has an impact on the normal T cell activation and proliferation in response to mitogens and also potentially antigen responsiveness.

Current Molecular Medicine, 2007

Heat shock proteins (hsps) are a highly conserved family of proteins, first recognized by their u... more Heat shock proteins (hsps) are a highly conserved family of proteins, first recognized by their upregulated expression in response to host exposure to raised temperatures. Further study has revealed that they have numerous functions in the cell, primarily as chaperones mediating both the correct folding of nascent polypeptide chains and the dissolution of aggregated protein complexes. The energy requirement for this chaperone activity is provided by the ATPase activity found in most families of hsps and thus the peptide binding capacity is controlled by ATP hydrolysis. The structural consequence of this is that hsps isolated in situ are found complexed to chaperoned peptides (hspCs). Much previous work has implicated hsps in the immune response to pathogens and recent studies have shown that the interaction of hsps with antigen presenting cells, such as dendritic cells (DCs), mediates the integration of the innate and acquired immune responses. This central role for hspCs in immunity is facilitated by their dual function in both innate immunity, with the induction of cytokines and the maturation of DCs mediated by the hsp component, and acquired immunity, with the trafficking of antigens chaperoned in hspCs for antigen presentation by the mature DCs.

Biochemical Society Transactions, 2004

The need for an effective TB (tuberculosis) vaccine remains acute, with tuberculosis still one of... more The need for an effective TB (tuberculosis) vaccine remains acute, with tuberculosis still one of the major killers worldwide and 3 million new infections annually. We report here on the immune responses elicited by HspCs (heat-shock protein–peptide complexes) isolated from BCG (Bacille Calmette–Guérin) vaccine. These HspCs elicit both the appropriate cellular and protective immune responses required to merit their further development as TB vaccine candidates.

The Journal of Infectious Diseases, 2012

Clinical and Experimental Immunology, 2004

SUMMARY The in -vivo clearance of Bordetella pertussis infections in murine models in naive mice ... more SUMMARY The in -vivo clearance of Bordetella pertussis infections in murine models in naive mice and animals vaccinated with whole-cell vaccine is considered to be via a Th-1-dependent mechanism in which interleukin-12 (IL)-12 may play a prominent role. It has also been demonstrated clearly that the treatment of animals with macrophage-derived IL-12 administered with an acellular vaccine can increase the efficacy of this vaccine preparation to levels seen with the whole-cell vaccine. However, the effects of exogenously added IL-12 on immune responses in non-vaccinated B. pertussis-challenged mice remain unclear, with two studies giving contradictory findings. In this study we have treated mice with escalating doses of mIL-12 (0·1–10 µg/mouse) prior to challenge with B. pertussis (using an aerosol challenge model of infection). The ability of mice to clear infection was assessed in IL-12 treated and in phosphate buffered saline (PBS) control animals at days 6 and 13 post-challenge. L...

BMC infectious diseases, Aug 12, 2016

In the absence of a validated animal model and/or an immune correlate which predict vaccine-media... more In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed. We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen. Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57...

This article cites 32 articles, 12 of which can be accessed free

Tuberculosis, 2016

The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brough... more The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brought together a wide and diverse community involved in tuberculosis vaccine research and development to discuss the current status of, and future directions for this critical effort. This paper summarizes the sessions on Low-Dose NHP Challenge Models, Novel Approaches to Animal Models for TB Vaccine R&D, Novel Antigen Delivery Strategies, and Next Generation TB Vaccines and Vaccine Concepts. Summaries of all sessions from the 4th Global Forum are compiled in a special supplement of Tuberculosis. [July 2016, Vol 99, Supp S1 - XX].