Megan Lloyd - Academia.edu (original) (raw)

Papers by Megan Lloyd

Research paper thumbnail of Immunization with Recombinant Murine Cytomegalovirus Expressing Murine Zona Pellucida 3 Causes Permanent Infertility in BALB/c Mice Due to Follicle Depletion and Ovulation Failure1

Biology of Reproduction, Nov 1, 2008

Zona pellucida (ZP) glycoproteins are promising candidate antigens for use in immunocontraceptive... more Zona pellucida (ZP) glycoproteins are promising candidate antigens for use in immunocontraceptive vaccines because of their crucial role in mammalian fertilization. A single intraperitoneal immunization with recombinant murine cytomegalovirus engineered to express murine ZP3 (rMCMV-mZP3) induces permanent infertility with no evident systemic illness in female BALB/c mice. To investigate the mechanisms underpinning reproductive failure elicited by rMCMV-mZP3, ovarian parameters and reproductive function were evaluated at time points spanning 10 days to 5 wk after virus inoculation. Fertility was substantially impaired by 14 days after inoculation with rMCMV-mZP3 and was fully ablated by 21 days. Pregnancies established after inoculation but before complete infertility showed no adverse effects on fetal viability assessed at Day 17.5 post coitum (pc). Infertile mice retained estrous cycling activity and remained receptive to mating; however, at Day 3.5 pc there were fewer developing embryos and corpora lutea, plasma progesterone content was reduced, and there was no evidence of excess unfertilized oocytes. Consistent with this, profound ovarian pathology was evident from 10 days after rMCMV-mZP3 inoculation, with a decline first in mature ovarian follicles and then in immature ovarian follicles and with diminished expression of genes regulating follicle development, including Nobox, Gdf 9, and Gja1 (connexin43). Follicle loss was associated with mild focal oophoritis and with recruitment of inflammatory leukocytes, predominantly CD4 + and CD8 + T cells evident from 10 days after virus inoculation. These data indicate that vaccination with rMCMV-mZP3 causes permanent infertility in BALB/c mice principally due to induction of ovarian autoimmune pathology leading to progressive oocyte depletion and eventual ovulation failure.

Research paper thumbnail of Innate and adaptive immune defence molecules and cells of human milk

European Journal of Immunology, Aug 1, 2016

Research paper thumbnail of 284. Leukocyte trafficking in the ovary of mice immunised with recombinant murine cyclomegalovirus expressing murine zona pellucida 3

Reproduction, Fertility and Development, 2005

Inoculation of female BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pe... more Inoculation of female BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pellucida antigen (MCMV-ZP3) confers infertility characterised by depletion in ovarian tertiary follicles by day 21 post inoculation followed by a progressive depletion in primordial follicles.1 Cell mediated immune responses begin as early as day 10 post immunisation with MCMV-ZP32 with the recruitment of leukocytes before serum antibody can be clearly detected in mice. The physiological mechanisms leading to infertility in inoculated mice are being progressively delineated with the role of leukocyte subsets implicated in early pathological changes in ovarian architecture. The aim of this study was to investigate the effect of MCMV-ZP3 infection on leukocytes including T cells recruited into the ovary following infection with recombinant virus. Fifteen BALB/c female mice were randomly allocated into three groups of five animals at 6 weeks of age. Group one received an injection of PBS, group two and three received intraperitoneal inoculations of 2 × 104 pfu of MCMV and MCMV-ZP3 respectively. Ovaries were retrieved at day 10, 21 and 35 post inoculation and one ovary from each mouse was sectioned for immunohistochemical analysis of resident leukocytes using mAb CD45 reactive with all leukocyte lineages and mAb for CD4 and CD8 positive T cells. MCMV-ZP3 inoculation increased the abundance of ovarian leukocytes including CD4 and CD8 positive T cells for all time points post immunisation except for CD8 positive T cells 21 days post infection (Table 1). These results suggest that leukocytes, including T cells, are involved in causing early changes in the ovary post infection with MCMV-ZP3 that lead to the depletion of existing ovarian follicles leading to life long infertility in mice. Further experiments are underway to investigate the role of antibody and changes in leukocyte populations in the ovary as the course of infection with recombinant virus progresses. This study is funded by the Cooperative Research Centre for Pest Animal Control. (1)Lloyd ML, et al. (2003). Biol. Reprod. 68, 2024–32.(2)O’Leary S, et al. (2004). Reprod. Fertil. Devel. 16(Supplement), 77.

Research paper thumbnail of Genetic variation in Australian spotted fever group rickettsiae

Journal of Clinical Microbiology, Jun 1, 1996

Research paper thumbnail of Characterization and comparison of Australian human spotted fever group rickettsiae

Journal of Clinical Microbiology, Nov 1, 1992

The microbiological and molecular characteristics of the rickettsiae isolated from humans with Qu... more The microbiological and molecular characteristics of the rickettsiae isolated from humans with Queensland tick typhus (QTT) caused by Rickettsia australis and the recently described Flinders Island spotted fever (FISF) were compared. Clinically and serologically, the diseases are similar. Cell culture reveals differences in the plaque-forming abilities of the isolates. Characterization of the gene encoding the genus-specific 17-kDa antigen of R. australis revealed a unique nucleotide sequence unlike those of the FISF isolate and Ricketfsia rickettsii. Southern blot analysis of rickettsial DNA from the isolates with a 17-kDa-antigen gene probe revealed the presence of this gene in all isolates but no difference in banding patterns. When a probe for the rRNA genes was used, clear differences in banding patterns of isolates from patients with QTT and FISF were revealed. Thus, the rickettsiae isolated from patients with FISF differ from those from patients with QTT and may represent a new rickettsial species.

Research paper thumbnail of The physiological consequences of exposure to zona pellucida 3 antibody in the mouse ovary

Journal of Reproductive Immunology, Aug 1, 2010

Research paper thumbnail of mouse sperm protein PH20

Assessment of contraceptive vaccines based on recombinant

Research paper thumbnail of Obituary: Geoff Shellam (1942–2015)

The Immunology and Virology communities lost a pioneer with the passing of Professor Geoffrey Ran... more The Immunology and Virology communities lost a pioneer with the passing of Professor Geoffrey Randolph Shellam on the 2nd of July...

Research paper thumbnail of Threshold learning outcomes for a microbiology major

Microbiology Australia, 2016

Research paper thumbnail of 222.Characterisation of the infertility effect induced by a recombinant murine cytomegalovirus expressing murine zona pellucida 3

Reproduction, Fertility and Development, 2004

Research paper thumbnail of Innate antiviral resistance influences the efficacy of a recombinant murine cytomegalovirus immunocontraceptive vaccine

Vaccine, 2007

Recombinant betaherpesviruses are attractive vaccine candidates because of their persistence in t... more Recombinant betaherpesviruses are attractive vaccine candidates because of their persistence in the host. A recombinant murine cytomegalovirus expressing the mouse ovarian glycoprotein zona pellucida 3 induces long lasting sterility in female BALB/c mice. Using inbred mouse strains selected for their innate resistance or susceptibility to MCMV, we show that genetically determined innate resistance to MCMV can reduce immunocontraceptive success. The Cmv1 locus that controls natural killer cell mediated responses to MCMV was implicated in determining vaccine efficacy. However, the role of the H-2 haplotype was less clear. Interestingly, Mus domesticus from an outbred colony of wild-derived mice were readily sterilised by vaccination, consistent with observations that strong innate immunity to MCMV is not common in Australian wild mice.

Research paper thumbnail of Characterization and comparison of Australian human spotted fever group rickettsiae

Journal of Clinical Microbiology, 1992

The microbiological and molecular characteristics of the rickettsiae isolated from humans with Qu... more The microbiological and molecular characteristics of the rickettsiae isolated from humans with Queensland tick typhus (QTT) caused by Rickettsia australis and the recently described Flinders Island spotted fever (FISF) were compared. Clinically and serologically, the diseases are similar. Cell culture reveals differences in the plaque-forming abilities of the isolates. Characterization of the gene encoding the genus-specific 17-kDa antigen of R. australis revealed a unique nucleotide sequence unlike those of the FISF isolate and Rickettsia rickettsii. Southern blot analysis of rickettsial DNA from the isolates with a 17-kDa-antigen gene probe revealed the presence of this gene in all isolates but no difference in banding patterns. When a probe for the rRNA genes was used, clear differences in banding patterns of isolates from patients with QTT and FISF were revealed. Thus, the rickettsiae isolated from patients with FISF differ from those from patients with QTT and may represent a n...

Research paper thumbnail of Geoff Shellam (1942–2015)

Immunology and Cell Biology, 2015

Research paper thumbnail of Prior infection with murine cytomegalovirus (MCMV) limits the immunocontraceptive effects of an MCMV vector expressing the mouse zona-pellucida-3 protein

Vaccine, 2008

We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zonapellu... more We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zonapellucida-3 gene (rMCMV-ZP3), which successfully induces infertility in experimentally inoculated laboratory or wild-derived mice. However, the future success of this vector as a fully disseminating vaccine in free-living mice may be compromised by pre-existing immunity since there is a high prevalence of naturally acquired MCMV infection in these mice. To evaluate the effect of prior immunity to MCMV on vaccine efficacy, we constructed two new biologically effective recombinant MCMV vectors expressing the mouse ZP3 protein from two MCMV strains (N1 and G4) derived from free-living mice. In wild mice, mixed MCMV infection is common and could be acquired either by simultaneous coinfection or sequential infection with different MCMV strains. Interestingly, while coinfection with both wild-type and rMCMV-ZP3 via the intraperitoneal route reduced the impact of the rMCMV-ZP3, prior infection with the same wild-type strain as that used to construct the rMCMV-ZP3 abrogated the immunocontraceptive effects of either N1-ZP3 or G4-ZP3. However, prior infection with G4 28 days before the introduction of N1-ZP3 had a reduced influence on the efficacy of the rMCMV-ZP3. Thus, the strain of virus and the timing of prior infection are factors that may influence the efficacy of the rMCMV-ZP3. Given that mixed infection of mice with MCMV is common, it is possible that prior immunity acquired by natural mucosal infection may have less a less inhibitory effect on the immunocontraceptive outcome.

Research paper thumbnail of Species-specificity of a murine immunocontraceptive utilising murine cytomegalovirus as a gene delivery vector

Vaccine, 2005

Cytomegaloviruses are species-specific DNA viruses. Recombinant murine cytomegaloviruse (MCMV) ex... more Cytomegaloviruses are species-specific DNA viruses. Recombinant murine cytomegaloviruse (MCMV) expressing the mouse egg-coat protein zona pellucida 3 (mZP3) has been shown to sterilise female mice by breaking self-tolerance and inducing an immune response against the host ZP3. This virus has the potential to be used for mouse population control, however the effect of this recombinant immunocontraceptive virus in non-host species must be determined. Recombinant MCMV-mZP3, based on both laboratory and wild strains of virus, induced long-lived antibody responses against structural viral proteins and mZP3 when inoculated into laboratory rats, although no viral DNA or replicating virus was identified. The anti-mZP3 antibodies were specific for mouse ZP3, did not cross-react with rat ZP3, and had no effect on the fertility of the rats.

Research paper thumbnail of Viral vectored immunocontraception: Screening of multiple fertility antigens using murine cytomegalovirus as a vaccine vector

Vaccine, 2007

Mouse cytomegalovirus (MCMV) has previously been used as a vaccine vector for viral vectored immu... more Mouse cytomegalovirus (MCMV) has previously been used as a vaccine vector for viral vectored immunocontraception (VVIC). MCMV expressing murine zona pellucida 3 (mZP3) induces long term infertility in up to 100% of female BALB/c mice following a single inoculation. Whilst a large number of antigens have been investigated as potential immunocontraceptive vaccines, it has been difficult to compare these antigens as few studies have used identical approaches or even animal species. Here a range of protein and polyepitope antigens, all expressed by MCMV, were tested for the ability to sterilise female mice. The antigens tested were bone morphogenic protein 15 (BMP15), oviduct glycoprotein (OGP) and ubiquitin-tagged mZP3. In addition, four polyepitope constructs that contain rodent or mouse specific epitopes were tested. This study found that when expressed by an MCMV vector, only full-length mZP3 or ubiquitin-tagged mZP3 induced infertility in female mice. BMP15 and OGP had no effect. Of the four polyepitopes tested, one had a partial effect on fertility. These data indicate that while MCMV is an effective vector for VVIC, the antigen used needs to be tested empirically. The partial infertility seen in mice infected with one of the polyepitope vaccines is a promising finding suggesting that it may be possible to combine a species specific virus with a species specific antigen for use as a disseminating mouse control agent.

Research paper thumbnail of Overcoming innate host resistance to vaccination: Employing a genetically distinct strain of murine cytomegalovirus avoids vector-mediated resistance to virally vectored immunocontraception

Vaccine, 2009

The laboratory strain of murine cytomegalovirus (MCMV), K181, has been successfully engineered as... more The laboratory strain of murine cytomegalovirus (MCMV), K181, has been successfully engineered as a vaccine expressing murine zona pellucida 3 (mZP3) for viral vectored immunocontraception (VVIC) in mice. However, certain laboratory strains of mice are resistant to infection with K181 and therefore demonstrate resistance to VVIC. Cmv1 is the best characterised innate resistance mechanism to MCMV and was first described in C57BL/6 mice. Resistance in C57BL/6 mice is due to early and strong activation of natural killer (NK) cells by an MCMV gene product, m157, that binds directly to the NK cell activating receptor Ly49H. In this study a wild strain of MCMV, G4, which expresses a variant m157 incapable of activating Ly49H, was engineered to express murine zona pellucida 3 (mZP3) and assessed for its ability to sterilise female C57BL/6 mice. When infected with K181-mZP3 female C57BL/6 mice remained fully fertile. In contrast, female C57BL/6 mice were sterilised by a single intraperitoneal inoculation of G4-mZP3. Infertility was induced by G4-mZP3 in three strains of mice that express Ly49H, on two different histocompatibility-2 (H-2) backgrounds. Finally, enhanced immunocontraception was observed in mice expressing H-2 k mediated resistance to MCMV when infected with G4-mZP3 compared to K181-mZP3. These data indicate that when using viral vaccine vectors, variant vector strains may be used to circumvent powerful innate immune responses against the vector and promote effective vaccination. This study highlights the importance of vaccine vector genetics in vaccination strategies.

Research paper thumbnail of Promoter control over foreign antigen expression in a murine cytomegalovirus vaccine vector

Vaccine, 2010

Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV)... more Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV) containing the mouse zona pellucida 3 (mZP3) gene for use as a virally vectored immunocontraceptive (VVIC). This study aimed to alter promoter control over foreign antigen expression and cellular localisation of the antigen expressed in order to overcome virus attenuation previously encountered. Early studies reported on the mZP3 gene expressed by a strong constitutive human cytomegalovirus immediate-early 1 promoter (pHCMV IE1). This virus was able to induce >90% infertility in BALB/c mice despite being heavily attenuated in vivo. In this study the mZP3 was placed under the control of the MCMV early 1 (pMCMV E1) promoter and the inducible tetracycline promoter (Tet-On). In both instances the recombinant virus was able to induce infertility in directly infected mice. However, the viruses remained attenuated. This study demonstrated the capacity to manipulate the nature of the immune response by altering promoter control over foreign antigen expression and cellular localisation of the expressed antigen. We were able to demonstrate that by using the MCMV E1 promoter it was still possible to sterilize female BALB/c mice with an MCMV vector expressing mZP3. The use of the MCMV E1 promoter provides an added level of safety to any MCMV based VVIC approach as it only allows for transgene expression in MCMV permissive cells.

Research paper thumbnail of Biological control of vertebrate pests using virally vectored immunocontraception

Journal of Reproductive Immunology, 2006

Species-specific viruses are being genetically engineered to produce contraceptive biological con... more Species-specific viruses are being genetically engineered to produce contraceptive biological controls for pest animals such as mice, rabbits and foxes. The virus vaccines are intended to trigger an autoimmune response in the target animals that interferes with their fertility in a process termed virally vectored immunocontraception. Laboratory experiments have shown that high levels of infertility can be induced in mice infected with recombinant murine cytomegalovirus and ectromelia virus expressing reproductive antigens as well as in rabbits using myxoma virus vectors. The strategies used to produce and deliver species-specific immunocontraceptive vaccines to free-living wildlife are presented in this review. Discussion includes coverage of the likely safety of the proposed vaccines as well as the implications of the approach for fertility control in other species.

Research paper thumbnail of The use of recombinant vaccines to induce effective contraception in mice

Journal of Reproductive Immunology, 2010

Research paper thumbnail of Immunization with Recombinant Murine Cytomegalovirus Expressing Murine Zona Pellucida 3 Causes Permanent Infertility in BALB/c Mice Due to Follicle Depletion and Ovulation Failure1

Biology of Reproduction, Nov 1, 2008

Zona pellucida (ZP) glycoproteins are promising candidate antigens for use in immunocontraceptive... more Zona pellucida (ZP) glycoproteins are promising candidate antigens for use in immunocontraceptive vaccines because of their crucial role in mammalian fertilization. A single intraperitoneal immunization with recombinant murine cytomegalovirus engineered to express murine ZP3 (rMCMV-mZP3) induces permanent infertility with no evident systemic illness in female BALB/c mice. To investigate the mechanisms underpinning reproductive failure elicited by rMCMV-mZP3, ovarian parameters and reproductive function were evaluated at time points spanning 10 days to 5 wk after virus inoculation. Fertility was substantially impaired by 14 days after inoculation with rMCMV-mZP3 and was fully ablated by 21 days. Pregnancies established after inoculation but before complete infertility showed no adverse effects on fetal viability assessed at Day 17.5 post coitum (pc). Infertile mice retained estrous cycling activity and remained receptive to mating; however, at Day 3.5 pc there were fewer developing embryos and corpora lutea, plasma progesterone content was reduced, and there was no evidence of excess unfertilized oocytes. Consistent with this, profound ovarian pathology was evident from 10 days after rMCMV-mZP3 inoculation, with a decline first in mature ovarian follicles and then in immature ovarian follicles and with diminished expression of genes regulating follicle development, including Nobox, Gdf 9, and Gja1 (connexin43). Follicle loss was associated with mild focal oophoritis and with recruitment of inflammatory leukocytes, predominantly CD4 + and CD8 + T cells evident from 10 days after virus inoculation. These data indicate that vaccination with rMCMV-mZP3 causes permanent infertility in BALB/c mice principally due to induction of ovarian autoimmune pathology leading to progressive oocyte depletion and eventual ovulation failure.

Research paper thumbnail of Innate and adaptive immune defence molecules and cells of human milk

European Journal of Immunology, Aug 1, 2016

Research paper thumbnail of 284. Leukocyte trafficking in the ovary of mice immunised with recombinant murine cyclomegalovirus expressing murine zona pellucida 3

Reproduction, Fertility and Development, 2005

Inoculation of female BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pe... more Inoculation of female BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pellucida antigen (MCMV-ZP3) confers infertility characterised by depletion in ovarian tertiary follicles by day 21 post inoculation followed by a progressive depletion in primordial follicles.1 Cell mediated immune responses begin as early as day 10 post immunisation with MCMV-ZP32 with the recruitment of leukocytes before serum antibody can be clearly detected in mice. The physiological mechanisms leading to infertility in inoculated mice are being progressively delineated with the role of leukocyte subsets implicated in early pathological changes in ovarian architecture. The aim of this study was to investigate the effect of MCMV-ZP3 infection on leukocytes including T cells recruited into the ovary following infection with recombinant virus. Fifteen BALB/c female mice were randomly allocated into three groups of five animals at 6 weeks of age. Group one received an injection of PBS, group two and three received intraperitoneal inoculations of 2 × 104 pfu of MCMV and MCMV-ZP3 respectively. Ovaries were retrieved at day 10, 21 and 35 post inoculation and one ovary from each mouse was sectioned for immunohistochemical analysis of resident leukocytes using mAb CD45 reactive with all leukocyte lineages and mAb for CD4 and CD8 positive T cells. MCMV-ZP3 inoculation increased the abundance of ovarian leukocytes including CD4 and CD8 positive T cells for all time points post immunisation except for CD8 positive T cells 21 days post infection (Table 1). These results suggest that leukocytes, including T cells, are involved in causing early changes in the ovary post infection with MCMV-ZP3 that lead to the depletion of existing ovarian follicles leading to life long infertility in mice. Further experiments are underway to investigate the role of antibody and changes in leukocyte populations in the ovary as the course of infection with recombinant virus progresses. This study is funded by the Cooperative Research Centre for Pest Animal Control. (1)Lloyd ML, et al. (2003). Biol. Reprod. 68, 2024–32.(2)O’Leary S, et al. (2004). Reprod. Fertil. Devel. 16(Supplement), 77.

Research paper thumbnail of Genetic variation in Australian spotted fever group rickettsiae

Journal of Clinical Microbiology, Jun 1, 1996

Research paper thumbnail of Characterization and comparison of Australian human spotted fever group rickettsiae

Journal of Clinical Microbiology, Nov 1, 1992

The microbiological and molecular characteristics of the rickettsiae isolated from humans with Qu... more The microbiological and molecular characteristics of the rickettsiae isolated from humans with Queensland tick typhus (QTT) caused by Rickettsia australis and the recently described Flinders Island spotted fever (FISF) were compared. Clinically and serologically, the diseases are similar. Cell culture reveals differences in the plaque-forming abilities of the isolates. Characterization of the gene encoding the genus-specific 17-kDa antigen of R. australis revealed a unique nucleotide sequence unlike those of the FISF isolate and Ricketfsia rickettsii. Southern blot analysis of rickettsial DNA from the isolates with a 17-kDa-antigen gene probe revealed the presence of this gene in all isolates but no difference in banding patterns. When a probe for the rRNA genes was used, clear differences in banding patterns of isolates from patients with QTT and FISF were revealed. Thus, the rickettsiae isolated from patients with FISF differ from those from patients with QTT and may represent a new rickettsial species.

Research paper thumbnail of The physiological consequences of exposure to zona pellucida 3 antibody in the mouse ovary

Journal of Reproductive Immunology, Aug 1, 2010

Research paper thumbnail of mouse sperm protein PH20

Assessment of contraceptive vaccines based on recombinant

Research paper thumbnail of Obituary: Geoff Shellam (1942–2015)

The Immunology and Virology communities lost a pioneer with the passing of Professor Geoffrey Ran... more The Immunology and Virology communities lost a pioneer with the passing of Professor Geoffrey Randolph Shellam on the 2nd of July...

Research paper thumbnail of Threshold learning outcomes for a microbiology major

Microbiology Australia, 2016

Research paper thumbnail of 222.Characterisation of the infertility effect induced by a recombinant murine cytomegalovirus expressing murine zona pellucida 3

Reproduction, Fertility and Development, 2004

Research paper thumbnail of Innate antiviral resistance influences the efficacy of a recombinant murine cytomegalovirus immunocontraceptive vaccine

Vaccine, 2007

Recombinant betaherpesviruses are attractive vaccine candidates because of their persistence in t... more Recombinant betaherpesviruses are attractive vaccine candidates because of their persistence in the host. A recombinant murine cytomegalovirus expressing the mouse ovarian glycoprotein zona pellucida 3 induces long lasting sterility in female BALB/c mice. Using inbred mouse strains selected for their innate resistance or susceptibility to MCMV, we show that genetically determined innate resistance to MCMV can reduce immunocontraceptive success. The Cmv1 locus that controls natural killer cell mediated responses to MCMV was implicated in determining vaccine efficacy. However, the role of the H-2 haplotype was less clear. Interestingly, Mus domesticus from an outbred colony of wild-derived mice were readily sterilised by vaccination, consistent with observations that strong innate immunity to MCMV is not common in Australian wild mice.

Research paper thumbnail of Characterization and comparison of Australian human spotted fever group rickettsiae

Journal of Clinical Microbiology, 1992

The microbiological and molecular characteristics of the rickettsiae isolated from humans with Qu... more The microbiological and molecular characteristics of the rickettsiae isolated from humans with Queensland tick typhus (QTT) caused by Rickettsia australis and the recently described Flinders Island spotted fever (FISF) were compared. Clinically and serologically, the diseases are similar. Cell culture reveals differences in the plaque-forming abilities of the isolates. Characterization of the gene encoding the genus-specific 17-kDa antigen of R. australis revealed a unique nucleotide sequence unlike those of the FISF isolate and Rickettsia rickettsii. Southern blot analysis of rickettsial DNA from the isolates with a 17-kDa-antigen gene probe revealed the presence of this gene in all isolates but no difference in banding patterns. When a probe for the rRNA genes was used, clear differences in banding patterns of isolates from patients with QTT and FISF were revealed. Thus, the rickettsiae isolated from patients with FISF differ from those from patients with QTT and may represent a n...

Research paper thumbnail of Geoff Shellam (1942–2015)

Immunology and Cell Biology, 2015

Research paper thumbnail of Prior infection with murine cytomegalovirus (MCMV) limits the immunocontraceptive effects of an MCMV vector expressing the mouse zona-pellucida-3 protein

Vaccine, 2008

We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zonapellu... more We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zonapellucida-3 gene (rMCMV-ZP3), which successfully induces infertility in experimentally inoculated laboratory or wild-derived mice. However, the future success of this vector as a fully disseminating vaccine in free-living mice may be compromised by pre-existing immunity since there is a high prevalence of naturally acquired MCMV infection in these mice. To evaluate the effect of prior immunity to MCMV on vaccine efficacy, we constructed two new biologically effective recombinant MCMV vectors expressing the mouse ZP3 protein from two MCMV strains (N1 and G4) derived from free-living mice. In wild mice, mixed MCMV infection is common and could be acquired either by simultaneous coinfection or sequential infection with different MCMV strains. Interestingly, while coinfection with both wild-type and rMCMV-ZP3 via the intraperitoneal route reduced the impact of the rMCMV-ZP3, prior infection with the same wild-type strain as that used to construct the rMCMV-ZP3 abrogated the immunocontraceptive effects of either N1-ZP3 or G4-ZP3. However, prior infection with G4 28 days before the introduction of N1-ZP3 had a reduced influence on the efficacy of the rMCMV-ZP3. Thus, the strain of virus and the timing of prior infection are factors that may influence the efficacy of the rMCMV-ZP3. Given that mixed infection of mice with MCMV is common, it is possible that prior immunity acquired by natural mucosal infection may have less a less inhibitory effect on the immunocontraceptive outcome.

Research paper thumbnail of Species-specificity of a murine immunocontraceptive utilising murine cytomegalovirus as a gene delivery vector

Vaccine, 2005

Cytomegaloviruses are species-specific DNA viruses. Recombinant murine cytomegaloviruse (MCMV) ex... more Cytomegaloviruses are species-specific DNA viruses. Recombinant murine cytomegaloviruse (MCMV) expressing the mouse egg-coat protein zona pellucida 3 (mZP3) has been shown to sterilise female mice by breaking self-tolerance and inducing an immune response against the host ZP3. This virus has the potential to be used for mouse population control, however the effect of this recombinant immunocontraceptive virus in non-host species must be determined. Recombinant MCMV-mZP3, based on both laboratory and wild strains of virus, induced long-lived antibody responses against structural viral proteins and mZP3 when inoculated into laboratory rats, although no viral DNA or replicating virus was identified. The anti-mZP3 antibodies were specific for mouse ZP3, did not cross-react with rat ZP3, and had no effect on the fertility of the rats.

Research paper thumbnail of Viral vectored immunocontraception: Screening of multiple fertility antigens using murine cytomegalovirus as a vaccine vector

Vaccine, 2007

Mouse cytomegalovirus (MCMV) has previously been used as a vaccine vector for viral vectored immu... more Mouse cytomegalovirus (MCMV) has previously been used as a vaccine vector for viral vectored immunocontraception (VVIC). MCMV expressing murine zona pellucida 3 (mZP3) induces long term infertility in up to 100% of female BALB/c mice following a single inoculation. Whilst a large number of antigens have been investigated as potential immunocontraceptive vaccines, it has been difficult to compare these antigens as few studies have used identical approaches or even animal species. Here a range of protein and polyepitope antigens, all expressed by MCMV, were tested for the ability to sterilise female mice. The antigens tested were bone morphogenic protein 15 (BMP15), oviduct glycoprotein (OGP) and ubiquitin-tagged mZP3. In addition, four polyepitope constructs that contain rodent or mouse specific epitopes were tested. This study found that when expressed by an MCMV vector, only full-length mZP3 or ubiquitin-tagged mZP3 induced infertility in female mice. BMP15 and OGP had no effect. Of the four polyepitopes tested, one had a partial effect on fertility. These data indicate that while MCMV is an effective vector for VVIC, the antigen used needs to be tested empirically. The partial infertility seen in mice infected with one of the polyepitope vaccines is a promising finding suggesting that it may be possible to combine a species specific virus with a species specific antigen for use as a disseminating mouse control agent.

Research paper thumbnail of Overcoming innate host resistance to vaccination: Employing a genetically distinct strain of murine cytomegalovirus avoids vector-mediated resistance to virally vectored immunocontraception

Vaccine, 2009

The laboratory strain of murine cytomegalovirus (MCMV), K181, has been successfully engineered as... more The laboratory strain of murine cytomegalovirus (MCMV), K181, has been successfully engineered as a vaccine expressing murine zona pellucida 3 (mZP3) for viral vectored immunocontraception (VVIC) in mice. However, certain laboratory strains of mice are resistant to infection with K181 and therefore demonstrate resistance to VVIC. Cmv1 is the best characterised innate resistance mechanism to MCMV and was first described in C57BL/6 mice. Resistance in C57BL/6 mice is due to early and strong activation of natural killer (NK) cells by an MCMV gene product, m157, that binds directly to the NK cell activating receptor Ly49H. In this study a wild strain of MCMV, G4, which expresses a variant m157 incapable of activating Ly49H, was engineered to express murine zona pellucida 3 (mZP3) and assessed for its ability to sterilise female C57BL/6 mice. When infected with K181-mZP3 female C57BL/6 mice remained fully fertile. In contrast, female C57BL/6 mice were sterilised by a single intraperitoneal inoculation of G4-mZP3. Infertility was induced by G4-mZP3 in three strains of mice that express Ly49H, on two different histocompatibility-2 (H-2) backgrounds. Finally, enhanced immunocontraception was observed in mice expressing H-2 k mediated resistance to MCMV when infected with G4-mZP3 compared to K181-mZP3. These data indicate that when using viral vaccine vectors, variant vector strains may be used to circumvent powerful innate immune responses against the vector and promote effective vaccination. This study highlights the importance of vaccine vector genetics in vaccination strategies.

Research paper thumbnail of Promoter control over foreign antigen expression in a murine cytomegalovirus vaccine vector

Vaccine, 2010

Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV)... more Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV) containing the mouse zona pellucida 3 (mZP3) gene for use as a virally vectored immunocontraceptive (VVIC). This study aimed to alter promoter control over foreign antigen expression and cellular localisation of the antigen expressed in order to overcome virus attenuation previously encountered. Early studies reported on the mZP3 gene expressed by a strong constitutive human cytomegalovirus immediate-early 1 promoter (pHCMV IE1). This virus was able to induce >90% infertility in BALB/c mice despite being heavily attenuated in vivo. In this study the mZP3 was placed under the control of the MCMV early 1 (pMCMV E1) promoter and the inducible tetracycline promoter (Tet-On). In both instances the recombinant virus was able to induce infertility in directly infected mice. However, the viruses remained attenuated. This study demonstrated the capacity to manipulate the nature of the immune response by altering promoter control over foreign antigen expression and cellular localisation of the expressed antigen. We were able to demonstrate that by using the MCMV E1 promoter it was still possible to sterilize female BALB/c mice with an MCMV vector expressing mZP3. The use of the MCMV E1 promoter provides an added level of safety to any MCMV based VVIC approach as it only allows for transgene expression in MCMV permissive cells.

Research paper thumbnail of Biological control of vertebrate pests using virally vectored immunocontraception

Journal of Reproductive Immunology, 2006

Species-specific viruses are being genetically engineered to produce contraceptive biological con... more Species-specific viruses are being genetically engineered to produce contraceptive biological controls for pest animals such as mice, rabbits and foxes. The virus vaccines are intended to trigger an autoimmune response in the target animals that interferes with their fertility in a process termed virally vectored immunocontraception. Laboratory experiments have shown that high levels of infertility can be induced in mice infected with recombinant murine cytomegalovirus and ectromelia virus expressing reproductive antigens as well as in rabbits using myxoma virus vectors. The strategies used to produce and deliver species-specific immunocontraceptive vaccines to free-living wildlife are presented in this review. Discussion includes coverage of the likely safety of the proposed vaccines as well as the implications of the approach for fertility control in other species.

Research paper thumbnail of The use of recombinant vaccines to induce effective contraception in mice

Journal of Reproductive Immunology, 2010