The aim of this study was to examine the effect of a glycoprotein B, gpB, vaccine in seronegative patients receiving kidneys from human Cytomegalovirus, HCMV, seropositive organ donors. Three seronegative patients were given a soluble gpB vaccine, while another three received placebos. Anti-gpB levels on the day of transplant were as much as 10 fold higher in gpB vaccinated patients compared to those who received placebos. Post-transplant serum viraemia of two gpB vaccinated patients increased at a slower rate and peaked at lower levels than those who received placebos. Decay rates and half-lives after initial viral load peaks were comparable among all patients.
HCMV is one of the most prevalent viruses with a worldwide infection rate of 60%-100% (ACOG 2000; Griffiths et al 2008). Hosts with a healthy immune system are generally asymptomatic but in the immunocompromised, it causes severe disease and has a formidable death rate. Vulnerable groups include neonates, transplant and AIDS patients. Transmission is through viral shedding at the cervix, semen, urine, saliva and breast milk (Ho 2008). Transplant patients are especially prone to infection through HCMV positive organs, (D+/R-), and their immunosuppressed state can bring about primary infection which causes life-threatening conditions for the patient (Ho 1991, 2001; Hendrix 1997).
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There is a 60% probability of HCMV infection through renal transplant, leading to a 90% chance of primary infection and a case fatality rate of 25% (Nunan & Banatvala 1984; Hecht et al 1988; van Son et al 1999). In D+/R- renal transplant patients, primary infection typically manifests itself in 60% to 100% of organ recipients, within the first 100 post-tranplant days. (Seale et al 2008; Humar et al 1999; Schroeder et al 2004). Without the control of viraemia, this can progress to debilitating disease such as CMV retinitis, allograft rejection and death. Before and in the early to mid 1980s, 100% of renal transplant associated mortality cases suffered from CMV pneumonia and 48% cardiac mortality was due to CMV related pulmonary involvement (Hecht et al 1988).
HCMV vaccine development has been ongoing and the approaches taken by researchers include attenuated virus, recombinant vector systems expressing envelope and core CMV proteins, T cell epitopes, and subunit vaccines (Arvin et al 2004; Schleiss 2008). This was a retrospective, placebo controlled study of effect of a soluble gpB subunit vaccine on D+/R- kidney transplant patients. GpB is a CMV envelope protein, localized on the surface of the virus and it mediates membrane fusion and viral entry into permissive cells. It also elicits effective CMV-specific, neutralizing antibodies (Arvin et al 2004; Schleiss 2008).
The six patients analysed were derived from a larger group of renal transplant patients who were HCMV seronegative recipients of kidneys from HCMV seropositive organ donors. Three were given pre-transplant vaccinations of a soluble gpB envelope subunit vaccine while the rest received placebos. Potentially neutralizing antibodies were measured in the vaccinated patients via an anti-gpB ELISA titre on the day of transplantation and compared with those patients who received placebos.
After receiving D+ kidneys, the HCMV viral loads of both patient groups were monitored by real-time PCR. Preemptive ganciclovir therapy was used to inhibit replicating virus when virus levels the sensitivity threshold, (â‰¥200 copies ml-1). Unfortunately, the viral loads of all six patients were not monitored for a uniform period of time and for the full 100 days after transplantation, in which primary infection usually occurs (Seale et al 2008; Humar et al 1999; Schroeder et al 2004). Placebo patients 1, 2 and 3 viral loads were monitored until days 124, 230 and 77 respectively after transplantation, while gpB vaccine patients 1, 2 and 3 were monitored until days 14, 38 and 58 respectively. Therefore comparative analyses between the two groups were limited up to 80 post-transplant days.
Pre-transplant antibody titres
All six patients had a detectable pre-transplant gpB-antibody titre, but it was markedly increased in the patients who received the soluble gpB subunit vaccine. Overall, the antibody titres of the vaccinated patients were higher in the vaccinated patients compared to those who received placebos. The average gpB antibody titre for gpB vaccinated patients was 2736, while the average titre for placebo recipients was 259.
Figure 1. HCMV gpB antibody titres, detected by ELISA. Placebo recipients, (Placebo pt. 1,2 and 3), have less HCMV antibodies detected in peripheral blood compared to the gpB vaccinated patients (Vaccine pt. 1,2 and 3).
Vaccine patient 3 had the highest titre level, 5250, followed by patients 1 and then 2. Vaccine patient 2 had a markedly lower titre (500) compared to vaccine patients 1 and 3. The placebo recipients 2 and 3 had roughly similar antibody titres, (305 & 323), while placebo patient 1 had a slightly lower titre of 150. (Refer to Figure 1 for ELISA titre results).
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Following kidney transplantation, the viral loads of all patients were measured using real-time PCR. The sensitivity threshold for quantifiable PCR detection was 200 copies ml-1. The baseline viral loads for placebo and gpB vaccinated patients were not above this threshold value and thus reported as 200 copies ml-1.
Growth rates, Doubling times, Peak viraemia
(GpB vaccinated Patient 1 was excluded from all analyses because viral loads were not measured past post-transplant day 14)
In placebo patients 1, 2 and 3, peripheral blood/serum viral loads begins to increase after post-transplant day 40, 30 and 20 respectively. Viral accumulation in peripheral blood increased at rates of 0.76, 0.42 and 0.5 copies ml-1 / day respectively, with an average doubling time of 1 day for all placebo recipients.
GpB vaccinated patients had slower growth rates 0.14 and 0.17 copies ml-1 / day, for patients 1 and 2 respectively and longer doubling time, ~4.5 days. (Refer to Figure 2 for growth rates & doubling times).
Preemptive ganciclovir therapy was administered to all patients when their viral load surpassed a 0.1 log10 increase in viraemia, (therapy initiation threshold). The placebo recipients' peaked at levels that were on average 12 times higher than the gpB vaccinated levels.
Decay rates, Half-lives of decline
With the exception of placebo patient 1, the decay rate of the other two placebo recipients was 0.23 copies ml-1 / day. This was slower than the decay rate of gpB vaccinated patients 1 and 2 at 0.53 and 0.41 copies ml-1 respectively. Therefore the took more time, 3 days, for the viral load to decay to half it's amount in placebo patients, compared to an average 1.5 days in gpB vaccinated patients.
Placebo patient 1 had a decay rate and half-life of viral decline that was similar to the gpB vaccinated patients, (0.53 copies ml-1 / day & 1.3 days respectively). (Refer to Figure 2 for viral decline rates & half-lives of decay).
Gpb is an ideal candidate for HCMV vaccine development because of it is highly conserved in all HCMV strains because of its infectivity function. This High conservation, combined with it being a major envelope glycoprotein also gives way for it to be the main target of neutralizing antibodies, nAbs. In fact, during the carouse of natural infection, GpB is thought to induce around 50% of all HCMV-specific nAbs (Britt & Fay et 1996; Marcoski et al 1996; Marshall & Stout et al 1994).
The increased abundance of gpB-specific antibodies, detected by ELISA in this experiment, seems to reflect the an upregulation of nAB production in gpB vaccinated patients. Vaccinated patient 2 elcited an antibody titre, much lower than the other 2 vaccinated patients, but the post-transplant viral dynamics suggests that this may not have made a difference.
Peripheral blood viraemic levels do not progress as quickly in vaccinated patients and peak at significantly lower levels compared to placebo patients. Patient 2's antibody profile and resultant viral dynamics further supports the idea that potency of these antibodies play a more significant role than abundance.
Upon ganciclovir administration, placebo patients do see suppression in viral load to â‰¤ the viral sensitivity threshold; the same as observed in gpB vaccinated patients, but there is a secondary replication reactivation / progression about 20 days after initial suppression with ganciclovir in two placebo patients. It may be safe to assume that if placebo patient 3's viral load was followed for more than 20 days after initial suppression, the same trend would be observed.
None of the vaccinated patients had their viral loads followed for more than 20 days after initial viral suppression. Therefore we cannot assume that there is no secondary progression of viral replication in vaccinated patients. Consequentially, it would have been beneficial to see if such an event still occurred in vaccinated patients, especially as their viral loads peaked at such relatively low levels.
The comparison of doubling times and half-lives of decay were promising in vaccinated patients because there seemed to be an overall inhibition in the rate of viral replication and it took a longer time for virus to increase by 50% to much lower levels than in placebos. Additionally, the response to ganciclovir in the vaccinated patients was approximately double that of the placebo patients with an expected increased half-life of decay. The importance of this lies in the correlation of viremic levels and the likelihood of disease. The probability and severity of HCMV disease increasing with each log10 increase of HCMV viral load past the viral threshold (Fox & Kidd 1995; Cope et al 1997).
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With regard to the efficacy of this vaccine and it adoption for use through the observations in this experiment, further correlation studies between these results and the disease outcomes of patients need to be done. Improvements to this study design to measure vaccine efficacy needed to extend to a consistency in the intervals and length of time in which viral loads were measured after transplantation and related immuno-pathogenesis needed to go past just a single ELISA done on the day of transplant.
The immunogenicity of the antibodies elicited is a crucial determinant of efficacy, but has not been addressed in this experiement. Assurance that the antibodies throughout the course of viral dynamics are derivatives of the vaccine as opposed to the immune response to transplant derived virus; and further more that these antibodies do in fact have potent neutralizing activities need to be illustrated. Therefore using only the parameters measured in this experiment, one can only recognize the potential of a gpB subunit vaccine as a possible barrier to infection and disease.
Previous human vaccine trials using gpB based vaccines have demonstrated the ability of this membrane anchored glycoproteins ability to induce mucosal IgA in addition to IgG (Wang & Adler et al 1996). This potential mucosal immunity is not seen with the live attenuated Towne vaccine or during the course of natural infection. Furthermore, GpB also has also shown to induce cytotoxic lymphocyte activity. Therefore the combination of both mucosal and systemic immunity could potentially prevent both infection and disease.
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