Graham Hall

Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques.

Prevention of sexually transmitted HIV infection was investigated in macaques by immunization with a recombinant SIV (simian immunodeficiency virus) envelope gp120 and core p27 vaccine. In two independent series of experiments, we used the novel targeted iliac lymph node (TILN) route of immunization, aiming close to the iliac lymph nodes draining the genitorectal mucosa. Rectal challenge with the SIVmac 32H J5 molecular clone in two series induced total protection in four out of seven macaques immunized by TILN, compared with infection in 13 of 14 unimmunized macaques or immunized by other routes (P = 0.025). The remaining three macaques showed either a decrease in viral load (>90%) or transient viremia, indicating that all seven TILN–immunized macaques showed total or partial protection (P = 0.001). Protection was associated with significant increase in the iliac lymph nodes of lgA antibody–secreting cells to p27 (P < 0.02), CD8–suppressor factor (P< 0.01), and the chemokines RANTES and MIP–1β (P< 0.01).

A Susceptible Mouse Model for Zika Virus Infection

Zika virus (ZIKV) is a mosquito-borne pathogen which has recently spread beyond Africa and into Pacific and South American regions. Despite first being detected in 1947, very little information is known about the virus, and its spread has been associated with increases in Guillain-Barre syndrome and microcephaly. There are currently no known vaccines or antivirals against ZIKV infection. Progress in assessing interventions will require the development of animal models to test efficacies; however, there are only limited reports on in vivo studies. The only susceptible murine models have involved intracerebral inoculations or juvenile animals, which do not replicate natural infection. Our report has studied the effect of ZIKV infection in type-I interferon receptor deficient (A129) mice and the parent strain (129Sv/Ev) after subcutaneous challenge in the lower leg to mimic a mosquito bite. A129 mice developed severe symptoms with widespread viral RNA detection in the blood, brain, spleen, liver and ovaries. Histological changes were also striking in these animals. 129Sv/Ev mice developed no clinical symptoms or histological changes, despite viral RNA being detectable in the blood, spleen and ovaries, albeit at lower levels than those seen in A129 mice. Our results identify A129 mice as being highly susceptible to ZIKV and thus A129 mice represent a suitable, and urgently required, small animal model for the testing of vaccines and antivirals.

Establishment of an Aerosol Challenge Model of Tuberculosis in Rhesus Macaques and an Evaluation of Endpoints for Vaccine Testing

ABSTRACT The establishment of an aerosol challenge model in nonhuman primates (NHPs) for the testing of vaccines against Mycobacterium tuberculosis would assist the global effort to optimize novel vaccination strategies. The endpoints used in preclinical challenge studies to identify measures of disease burden need to be accurate and sensitive enough to distinguish subtle differences and benefits afforded by different tuberculosis (TB) vaccine regimens when group sizes are inevitably small. This study sought to assess clinical and nonclinical endpoints as potentially sensitive measures of disease burden in a challenge study with rhesus macaques by using a new protocol of aerosol administration of M. tuberculosis. Immunological and clinical readouts were assessed for utility in vaccine evaluation studies. This is the first example of TB vaccine evaluation with rhesus macaques where long-term survival was one of the primary endpoints. However, we found that in NHP vaccine efficacy studies with maximum group sizes of six animals, survival did not provide a valuable endpoint. Two approaches used in human clinical trials for the evaluation of the gamma interferon (IFN-γ) response to vaccination (enzyme-linked immunospot [ELISpot] assay and enzyme-linked immunosorbent assay [ELISA]) were included in this study. The IFN-γ profiles induced following vaccination were found not to correlate with protection, nor did the level of purified protein derivative (PPD)-specific proliferation. The only readout to reliably distinguish vaccinated and unvaccinated NHPs was the determination of lung lesion burden using magnetic resonance (MR) imaging combined with stereology at the end of the study. Therefore, the currently proposed key markers were not shown to correlate with protection, and only imaging offered a potentially reliable correlate.

The role of γ δ T cells in generating antiviral factors and β‐chemokines in protection against mucosal simian immunodeficiency virus infection

In view of the role of γ δ+ T cells in mucosal protection against infection, the proportion of γ δ T cells was examined in cells eluted from lymphoid and mucosal tissues of macaques immunized with simian immunodeficiency virus (SIV) gp120 and p27 in alum and challenged with live SIV by the rectal mucosal route. This revealed a significant increase in γ δ T cells eluted from the rectal mucosa (p < 0.01) and the related iliac lymph nodes (p < 0.0001) in protected as compared with infected macaques. Preferential homing of PKH‐26‐labeled γ δ+ T cells from the primed iliac lymph nodes to the rectal and cervico‐vaginal mucosa was demonstrated after targeted iliac lymph node as compared with i. m. immunization. Investigations of the mechanism of protection revealed that γ δ+ T cells can generate antiviral factors, RANTES, macrophage inflammatory protein (MIP)‐1α and MIP‐1β which can prevent SIV infection by binding to the CCR5 coreceptors. Up‐regulation of γ δ+ T cells was demonstrated by immunization of macaques with heat shock protein (HSP)70 linked to peptides and with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). This was confirmed by in vitro studies showing that GM‐CSF can up‐regulate γ δ+ T cells from macaques immunized with HSP‐linked peptides but not those from naive animals. We suggest that a novel strategy of immunization with HSP70 linked to antigen may generate both cognate immunity to the antigen and innate immunity by virtue of up‐regulation of γ δ+ T cells. These cells generate antiviral factors and the three β‐chemokines that prevent binding and transmission of SIV or M‐tropic HIV by the CCR5 coreceptor.

Mycobacterium tuberculosis arabinomannan–protein conjugates protect against tuberculosis

Lipoarabinomannan (LAM) is a major structural surface component of mycobacteria. Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen. The different AM oligosaccharide (AMOs)-protein conjugate vaccine candidates proved to be highly immunogenic, inducing boosterable IgG responses against the AMOs portion of the conjugates in rabbits and guinea-pigs. Proliferation of T-cells from C57BL/6 mice immunized with the conjugates was seen upon in vitro stimulation with PPD. In C57BL/6 mice subcutaneous immunization with the AMOs-antigen 85B conjugate in alum provided significant protection compared to sham (alum only) immunized mice (P < 0.021) as estimated by long term survival against intravenous challenge with 10(5) M. tuberculosis H37Rv. Subcutaneous immunization followed by nasal boost with an AMOs-TT conjugate in Eurocine L3 adjuvant provided high (P < 0.025) protection as determined by long term survival after intranasal challenge with 10(5) virulent M. tuberculosis strain Harlingen. This level of protection was comparable to that obtained with the conventional live attenuated BCG vaccine. In guinea-pigs, immunization with AMOs-Ag85B in Eurocine L3 adjuvant followed by aerogenic challenge with M. tuberculosis H37Rv resulted in increased survival and reduced pathology in lungs and spleens relative to non-immunized animals.

Identification of a Mycobacterium bovis BCG Auxotrophic Mutant That Protects Guinea Pigs against M. bovis and Hematogenous Spread of Mycobacterium tuberculosis without Sensitization to Tuberculin

ABSTRACT Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.

Vaccination of Guinea Pigs with DNA Encoding the Mycobacterial Antigen MPB83 Influences Pulmonary Pathology but Not Hematogenous Spread following Aerogenic Infection with Mycobacterium bovis

ABSTRACT Protection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistent Mycobacterium bovis infection in wildlife and in developing countries where it is not economical to implement a tuberculin test and slaughter control program. The main aim of such a vaccination strategy would be to reduce transmission of infection by reducing the lung pathology caused by infection and preventing seeding of the organism to organs from which M. bovis could be excreted. Recent reports of successful DNA vaccination against Mycobacterium tuberculosis in small-animal models have suggested that DNA vaccines act by reducing lung pathology without sensitizing animals to tuberculin testing. We therefore evaluated the ability of vaccines consisting of DNA encoding the mycobacterial antigens MPB83 and 85A to reduce lung pathology and prevent hematogenous spread in guinea pigs challenged with a low dose of aerosolized M. bovis. Vaccination with MPB83 DNA reduced the severity of pulmonary lesions, as assessed by histopathology, and resembled M. bovis BCG vaccination in this respect. However, unlike BCG vaccination, MPB83 DNA vaccination did not protect challenged guinea pigs from hematogenous spread of organisms to the spleen. In contrast, vaccination with antigen 85A DNA, a promising DNA vaccine for human tuberculosis, had no measurable protective effect against infection with M. bovis.

Up-regulation of beta-chemokines and down-modulation of CCR5 co-receptors inhibit simian immunodeficiency virus transmission in non-human primates.

A non‐cognate mechanism of protection against human immunodeficiency virus‐1 (HIV‐1) infection involves up‐regulation of β‐chemokines, which bind and may down‐modulate the CCR5 co‐receptors, thereby preventing transmission of M‐tropic HIV‐1. The objective of this investigation was to evaluate this mechanism in vivo in non‐human primates. Rhesus macaques were immunized by a modified targeted lymph nodes (TLN) route with recombinant simian immunodeficiency virus (SIV) glycoprotein 120 (gp120) and p27 in alum, and adsorbed recombinant granulocyte–macrophage colony‐stimulating factor (GM‐CSF) with either interleukin (IL)‐2 or IL‐4. Immunization induced significant increases in the concentrations of CD8 cell‐derived suppressor factor (CD8‐SF), regulated on activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)‐1α and MIP‐1β, and down‐modulation of the proportion of cells expressing CCR5 (r = 0·737, P < 0·05). The macaques were then challenged with SIVmac 220 by the rectal mucosal route. The plasma SIVmac RNA showed a significant inverse correlation with the CD8‐SF or the concentration of the three β‐chemokines (r = 0·831 and 0·824, P < 0·01), but a positive correlation between the proportion of CCR5+ cells and SIVmac RNA (r = 0·613, P = 0·05). These results demonstrate for the first time in vivo that immunization up‐regulates β‐chemokines, which may down‐modulate CCR5 co‐receptors, and both functions are significantly correlated with the viral load. Hence, the non‐cognate β‐chemokine–CCR5 mechanism should be considered as complementary to specific immunity in vaccination against HIV.

A guinea pig model of low-dose Mycobacterium bovis aerogenic infection

In order to develop a model of Mycobacterium bovis infection with pathogenetical relevance, a modified version of the Henderson apparatus was used to deliver infectious aerosols directly to the snouts of guinea pigs. Aerosols generated from 10(6), 10(7), 10(8)CFU/ml M. bovis suspensions established disease in every animal, with estimated retained doses of 10, 100, 1000 CFU, respectively. For comparison, other guinea pigs were inoculated with 100 CFU M. bovis intramuscularly (i.m.). Pathology and bacterial colonisation of lungs and spleen varied according to the dose and route of inoculation. Animals inoculated i.m. gave a significant cutaneous tuberculin hypersensitivity reaction earlier after testing than those infected aerogenically. A serological response to M. bovis antigens was detected in all infected animals. Intensity of antigen recognition was dose-dependent and although the range of antigens recognised varied between animals, a 25 kDa antigen present in the cell fraction was serodominant. Thus, a reproducible guinea pig model has been defined that may be suitable for virulence, vaccination, and immunological studies.

In vivo resistance to simian immunodeficiency virus superinfection depends on attenuated virus dose.

Infection of macaques with attenuated simian immunodeficiency virus (SIV) induces potent superinfection resistance that may be applicable to the development of an AIDS vaccine but little information exists concerning the conditions necessary for the induction of this vaccine effect. We report that only a high dose of attenuated SIVmac protected macaques against intravenous challenge with more virulent virus 15 weeks after primary infection. Three of four animals given 2000-20000 TCID50 of SIVmacC8, a molecular clone of SIVmac251(32H) with a 12 bp deletion in the nef gene, essentially resisted superinfection with uncloned SIVmac. In two animals challenge virus was never detected by PCR and in one animal challenge virus was detected on one occasion only. Although animals given 2-200 TCID50 of attenuated virus were superinfected they were spared from the loss of CD4 cells seen in infected naive controls. Protection from superinfection did not correlate with immune responses, including the levels of virus-specific antibodies or virus-neutralizing activity measured on the day of challenge; although, after superinfection challenge, Nef-specific CTL responses were detected only in animals infected with high doses of attenuated SIV. Unexpectedly, cell-associated virus loads 2 weeks after inoculation were significantly lower in animals infected with a high dose of attenuated SIV compared to those in animals infected with a low dose. Our results suggest that the early dynamics of infection with attenuated virus influence superinfection resistance.