VACCINE COMPOSITIONS

Abstract

The invention relates to vaccines for the prevention or treatment of infectious diseases, and to methods of preparing or delivering such vaccines. In particular, a vaccine for use in the prevention or treatment of disease is used in a dose of, via a parenteral route without adjuvant less than 0.03 .Math.g antigen and with adjuvant less than 0.003 .Math.g antigen, and via a mucosal route without adjuvant less than 1 .Math.g antigen and/or the equivalent of 1.6 × 10.sup.7 PFU and with adjuvant less than 0.04 .Math.g antigen and/or the equivalent of 1.6 × 10.sup.7 PFU

Claims

1. A method for the prevention or treatment of a disease in a subject comprising administering an effective amount of a vaccine to the subject wherein the vaccine is not a live attenuated vaccine and wherein the vaccine is used in a dose of, via a parenteral route without adjuvant less than 0.03 .Math.g antigen and with adjuvant less than 0.003 .Math.g antigen, and via a mucosal route without adjuvant less than 1 .Math.g antigen and with adjuvant less than 0.04 .Math.g antigen.

2. The method according to claim 1, wherein the vaccine is used to prevent or treat a respiratory infection and/or airborne infection.

3. The method according to claim 1, wherein the vaccine is administered to the respiratory tract.

4. The method according to claim 1, wherein the vaccine is administered to the lungs.

5. The method according to claim 1, wherein the disease is A a respiratory or airborne infection, wherein the vaccine is delivered to the lungs of the subject to be treated and wherein the vaccine is administered in a dose of less than 0.003 .Math.g.

6. The method according to claim 1, wherein the dose is less than 0.001 .Math.g.

7. The method according to claim 1, wherein the dose is less than 0.0003 .Math.g.

8. (canceled)

9. The method according to claim 1, wherein the infection is a viral infection.

10. The method according to claim 9, wherein the vaccine is or comprises an inactivated virus.

11. The method according to claim 1, wherein the vaccine is a vaccine against influenza, tuberculosis, MERS, SARS, rhinovirus, measles, Ebola, Chlamydia pneumonia, respiratory syncytial virus, pneumococci or FMDV.

12. (canceled)

13. The method according to claim 1, wherein the method comprises producing said vaccine composition and administering it to the lungs of a subject.

14. The method according to claim 13, wherein said method comprises inactivating the infectious agent responsible for said disease and delivering the inactivated agent to the lungs of the subject.

15. The method according to claim 13, wherein the vaccine is made in situ using a portable device.

16. (canceled)

17. (canceled)

18. (canceled)

19. The method according to claim 1, wherein the vaccine is delivered to the subject using a device comprising a nebulizer.

20. The method according to claim 1, wherein the vaccine is delivered as and/or comprises an aerosol.

21. The method according to claim 1, wherein the vaccine does not comprise or is not co-administered with an adjuvant.

22. The method according to claim 1, wherein (a) the subject to be treated has already been primed by exposure to the same, or similar, pathogen and/or has been vaccinated with a vaccine for the same or similar pathogen; or (b) the vaccine is delivered in an initial priming dose followed by a boost; or (c) the vaccine is delivered on 3 or more occasions to a subject.

23. (canceled)

24. (canceled)

25. The method according to claim 1, wherein the disease is an infectious disease, wherein the effective amount is less than 0.003 .Math.g antigen .

26. The method according to claim 3, wherein (i) the vaccine is made in situ by inactivating live virus in the air, or (ii) the vaccine is made in situ by inactivating live virus in the air wherein said method is carried out in an indoor environment or in a mode of transport.

27. A method of delivering a vaccine to a human subject at risk of developing a viral infection, wherein the vaccine is a vaccine against said virus that comprises inactivated virus, and is not a live attenuated vaccine, and wherein the vaccine is delivered via a mucosal route in a dose of, without adjuvant less than 1 .Math.g antigen, or with adjuvant less than 0.04 .Math.g antigen, optionally wherein said method comprises inactivating said virus and delivering the inactivated virus to the lungs of the human subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0143] The invention is further described by way of example only with reference to the attached figures:

[0144] FIG. 1. Study schematic. Upward pointing arrows show days of nasal wash sampling; (S) show days of serum sampling. Days are numbered relative to the H3N2 priming infection on T=0.

[0145] FIG. 2. Serum HAI titres against H3N2 virus for Groups 1-4. Points represent geometric mean titre for each group

[0146] FIG. 3. Serum HAI titres against H1N1 virus. Points represent geometric mean titre for each group. Titres of <4 are plotted as 2 to allow visualisation.

[0147] FIG. 4. Group mean nasal wash cell counts. Points represent the geometric mean titre for each group.

[0148] FIG. 5. Group geometric mean nasal wash titres following H1N1 challenge on day 56. Samples with no plaques detected were plotted as 1 PFU/ml.

[0149] FIG. 6. Weight change following H3N2 virus challenge. Points represent group means.

[0150] FIG. 7. Weight change following H1N1 aerosol spray. Points represent group means.

[0151] FIG. 8. Weight change following H1N1 intra-nasal challenge. Points represent group means.

EXAMPLE 1

[0152] When infected by influenza, ferrets share symptoms that are very similar to human influenza. For this and other reasons ferrets are therefore considered perhaps the best animal model for human influenza. Accordingly, ferrets were used to test immunisation with very low doses of influenza. Manufactured vaccine preparation, in the form of formalin killed virus, and in situ vaccine, in the form of live virus passed through a UV sterilising device as described in patent application WO2008/120005, were used. Animals were pre-infected on day 0 with H3N2 virus (Influenza A/Perth/16/09) to mimic the natural situation where most people have already been infected with one or more stains of influenza and/or been vaccinated against seasonal influenza. The immunising and challenge virus was an H1N1 virus (Influenza A/California/04/09).

Overall Experimental Design

[0153] All ferrets were primed by infection with 100 PFU H3N2 virus delivered i.n. at T=0. After 28 days, ferrets were divided into 4 groups for aerosol spray:

TABLE-US-00001 Study groups. Group Aerosol virus Days of aerosol spray 1 H1N1 untreated T+28 only 2 H1N1, UV-inactivated T+28, 38, 47 3 H1N1, formalin-inactivated T+28, 38, 47 4 Mock (PBS) T+28 only

[0154] H1N1 untreated animals were exposed to H1N1 virus through the sterilisation device but with the UV light off. Therefore, the animals were exposed to live virus.

[0155] H1N1, UV-inactivated animals were exposed to H1N1 virus through the sterilisation device but with the UV light on (dose 600 Jm.sup.-2 UV delivered). Therefore, the animals were exposed to UV-inactivated virus.

[0156] H1N1, formalin-inactivated animals were exposed to H1N1 virus that had been inactivated with formalin through the sterilisation device but with the UV light off. Therefore, the animals were exposed to formalin-inactivated virus.

[0157] Mock (PBS) animals were exposed to PBS through the sterilisation device with the light off. Therefore, the animals were an un-immunised control group, not exposed to virus.

[0158] All aerosol immunising doses were of 500 PFU delivered by aerosol to the lungs. The nebuliser delivered particles of count median aerodynamic diameter 0.72 - 0.78 .Math.m, mass median aerodynamic diameter 1.3 - 1.5 .Math.m.

[0159] On day T+56, all ferrets were challenged with a low dose (100 PFU) of H1N1 virus i.n.

Aerosol Infection of Ferrets

[0160] Two 5 minute sprays were conducted for each group, 3 ferrets per spray, using the sterilisation device connected to a 6-jet Collison nebuliser, in the order groups 4, 3, 2, 1. On days T+38 and T+47, only groups 2 and 3 were sprayed.

[0161] A previous study conducted at Public Health England (PHE) Porton using the Collison nebuliser and Henderson apparatus showed that the presence of the air sterilisation device (with UV light set to off) had no measurable effect on the spray factor of the H1N1 virus. Using the known mean spray factor of 1.53 × 10.sup.-6, and a mean weight (measured on T+26) for the ferrets of 0.9066 kg, it was calculated that a nebuliser concentration of 1.88 × 10.sup.5 PFU/ml was required to give a presented dose of 500 PFU to each ferret. For formalin-fixed virus, a dilution was used based on the recovery in HAU, and the measured PFU/HAU ratio a ratio of 7.8 × 10.sup.4 PFU/HAU for the starting material. Thus, on days 28, 38 and 47 the immunising dose was 500 PFU of virus.

Results

Priming of Ferrets With H3N2 Virus

[0162] Ferrets in all groups were infected intra-nasally with 100 PFU H3N2 virus on T=0. Back-titration of the virus inoculum gave a titre of 375 PFU/ml, which is within 2-fold of the target titre of 500 PFU/ml. Successful infection of all ferrets was confirmed by a rise in nasal wash cell counts on T+4 (see FIG. 4), and rise in haemagglutination inhibition (HAI) titre on T+28 (FIG. 2). For the 6 ferrets showing the lowest rises in nasal wash cell count, plaque assays were performed on the nasal wash fluid, and confirmed all 6 ferrets were actively shedding virus.

Serum HAI Titres

[0163] All sera were treated with 3 volumes of receptor destroying enzyme (RDE) prior to titration to remove any non-specific inhibitors of haemagglutination. Sera from days T=-3 (pre-bleed), T+28 and T+70 were titrated against H3N2 (A/Perth/16/09) and H1N1 (A/California/07/09, antigenically indistinguishable from A/California/04/09) viruses using chicken red blood cells. In addition, sera taken at T+38, T+47 and T+56 were titrated against H1N1 virus.

[0164] All titres against both viruses on T=-3 were ≤ 8, and so considered to be sero-negative. All ferrets showed sero-conversion to H3N2, but not H1N1, virus by T+28 (titres ≥ 320). H3N2 titres remained high (≥ 160) until the end of the study at T+70 (FIG. 2).

[0165] Only group 1 sero-converted to H1N1 virus by 10 days after the aerosol spray on T+28. Groups 2, 3 and 4 sero-converted to H1N1 virus by 14 days after the intra-nasal H1N1 challenge on T+56 (FIG. 3).

Nasal Wash HAI Titres

[0166] Nasal wash fluids taken on T+38 and T+56 were titrated by HAI without prior RDE-treatment, starting from a 2-fold dilution. All titres were ≤ 2 on both days. As these days were presumed to be the most likely to show a mucosal immune response, nasal wash fluids from other days were not titrated.

Nasal Wash Cell Counts

[0167] Counts of viable cells in nasal wash fluid typically rise from ≤ 10.sup.5 cells/ml to 10.sup.6-10.sup.7 cells/ml a few days after virus infection. This rise is a consequence of the innate immune response to infection.

[0168] The rise in counts of all groups at T+4 represents the immune response to the H3N2 infection at T=0 (FIG. 4). Cell counts then fell to baseline levels prior to the aerosol infection on T+28. Following aerosol sprays, only group 1 show a rise in cell counts 3-5 days later, suggesting that the UV-treated and formalin-treated viruses were unable to initiate infection of the ferrets. Groups 2-4 show a rise in cell counts 3-7 days after H1N1 virus challenge on T+56, whereas group 1 did not show a rise.

Titration of Aerosol-Sprayed Virus by Plaque Assay

[0169] In order to estimate presented doses of aerosolised virus, remaining nebuliser fluid and collected impinger fluid were titrated by plaque assay. As formalin-fixed virus and virus which had passed through the influenza aerosol sterilising device (IASD) with the UV light on, were expected to show no infectivity, samples of nebuliser and impinger fluids were extracted for RNA and titrated by real-time reverse transcription-polymerase chain reaction (RT-PCR).

TABLE-US-00002 Plaque assay titres of nebuliser and impinger fluids. n/c, plaques not countable. Titres in PFU/ml, mean of 2 replicates. *Two plaques in one well of one replicate. T+28 T+38 T+47 Group nebuliser impinger nebuliser impinger nebuliser impinger 1 n/c n/c 2 n/c 0 2.63×10.sup.5 0 1.03×10.sup.5 2.5* 3 0 0 0 0 0 0 4 0 0

[0170] Due to technical problems with the Madin-Darby Canine Kidney (MDCK) cells it was not possible to obtain accurate titres for the T+28 samples. Infection was apparent (in n/c groups), but due to problems with the monolayer plaques could not be counted accurately. The group 2 nebuliser titres on T+38 and T+47 were within 2-fold of the target titre of 1.88 × 10.sup.5 PFU/ml. The same dilution of the same virus stock was used in the nebuliser for groups 1 and 2 on T+28, as for group 2 on T+38 and T+47. The spray factor calculated from RT-PCR titres for Group 1 was very close to the expected value, supporting the conclusion that the target presented dose of 500 PFU per ferret had been achieved. RT-PCR also confirmed that the dose of virus delivered on day T+28 was comparable to those delivered on days T+38 and T+47. No live virus was detected in the formalin-fixed virus group 3. No infectivity was detected in the impinger following UV-treatment of the virus for group 2 at T+28 and T+38. The low titre of 2.5 PFU/ml (calculated from 2 plaques in a single well) seen in the impinger for group 2 at T+47 was only detected in one impinger replicate. As none of the group 2 ferrets showed any signs of infection between days T+47 and T+56, it is assumed that the plaques were the result of contamination, either during collection of the impinger fluids or during set-up of the plaque assays.

Virus Titrations

[0171] Nasal washes from groups 1-3 for T+31 (3 days after first aerosol sprays) were titrated to confirm infection of ferrets by the aerosol route (Table 3).

TABLE-US-00003 Plaque titration of T+31 nasal washes. Animal ID Plaques per ml Animal ID Plaques per ml Animal ID Plaques per ml Group 1 Group 2 Group 3 25004 3.50×10.sup.3 24989 0 24779 0 25005 6.75×10.sup.4 24993 0 24784 0 25002 3.50×10.sup.5 24992 0 24777 0 24998 3.75×10.sup.3 24995 0 24778 0 24996 2.50×10.sup.4 24782 0 24781 0 25000 3.50×10.sup.3 24783 0 24994 0

[0172] All 6 ferrets in group 1 (infected with UV light off) were infected and shedding virus. No virus was detected in nasal wash from ferrets of groups 2 (UV light on) or 3 (formalin-fixed).

Nasal Wash Cell Counts Following Challenge

[0173] Following H1N1 intra-nasal challenge on T+56, nasal washes were collected and titrated 1, 3, 5 and 7 days later (FIG. 5).

Virus Titrations Following Challenge

[0174] No virus was detected in nasal washes from group 1 at any time-point. All 18 ferrets in groups 2-4 showed virus in nasal washes, peaking 3 days post-challenge. Titres were not significantly different between groups 2-4 on days T+57, T+59 or T+61 (1-way ANOVA). Area under the curve was calculated for each ferret, there was no significant difference between groups 2-4 (1-way ANOVA, p = 0.20).

Clinical Signs of Infection

[0175] Bodyweights were measured daily, except for days T+43-55 inclusive.

[0176] The H3N2 virus challenge on T=0 resulted in minor weight loss, with each group showing a dip (1.7 % drop in 1 day) in mean weight on T+3 (FIG. 6).

[0177] Other clinical signs of infection (sneezing, nasal discharge and/or loss of appetite) were observed in all groups following the H3N2 challenge, mostly between days 5 and 11 post-infection. No instances of inactivity or diarrhoea were observed in the 28 days post-infection.

[0178] Following the first aerosol sprays on T+28, only group 1 showed weight loss (FIG. 7).

[0179] Only group 1 showed any other clinical signs following aerosol spray challenge, namely 5 instances of sneezing. No instances of nasal discharge, inactivity, loss of appetite or diarrhoea were observed in any of the ferrets between days T+28 and T+42.

[0180] Following the H1N1 intra-nasal challenge on T+56, significant weight loss was observed in group 4 (mock-sprayed) relative to group 1 (sprayed with infectious virus) (t-test, p < 0.0001 on T+59) (FIG. 8). Maximum mean weight loss of ~10 % (group 4) was in line with previous studies using low-dose intra-nasal H1N1 challenge.

[0181] Groups 2 and 3 showed an intermediate level of weight loss relative to groups 1 and 4. Group 2 weights were significantly less than group 1 on days T+58-66 and T+68-70 (t-tests, p < 0.05)(12 out of 14 days). Group 2 weights were significantly greater than group 4 on days T+57-59 and T+61-70 (t-tests, p < 0.05), i.e. 13 out of 14 days.

[0182] In order to compare groups across days T+56 to 70 inclusive, weight gain or loss relative to T+56 (i.e. with T+56 set to 0 %) was plotted and area under the curve was calculated for each ferret. Then groups were compared by 1-tailed t-test:

TABLE-US-00004 Comparison of areas under the curves for weight loss between groups. The p-values for each pair of groups are shown. Group 2 3 4 1 0.007 0.002 0.001 2 0.432 0.022 3 0.024

[0183] All groups showed significantly different weight loss from each other, except groups 2 and 3 were not significantly different (Table 4).

[0184] There appeared to be no significant difference in observations of sneezing, nasal discharge and loss of appetite between groups 2, 3 and 4 for days T+56 to T+70, although all appeared greater than for group 1.

Conclusions

[0185] H3N2 priming was confirmed by the rise in nasal wash cell counts in all groups, and the high H3N2-specific HAI serum titres seen at T+28. The sprays on T+28 resulted in infection of all ferrets in group 1 (as expected for infectious virus), but no ferrets in groups 2-4, confirming that UV-treatment and formalin treatment had ablated the infectivity of the virus inocula. The spray factor calculated from the RT-PCR titres for group 1 was very close to the expected value, confirming the target presented dose of 500 PFU per ferret, and this was supported by the plaque assay data. Group 1 showed clear protection from the H1N1 virus challenge on T+56: no weight loss, minimal clinical signs, no rise in nasal wash cell count, and no detectable virus in nasal washes. This protection was correlated to the high H1N1-specific serum HAI titre observed at T+56. Groups 2 and 3 showed no detectable HAI titres on T+38 and T+56 in either serum or nasal wash. Groups 2 and 3 were not protected against infection per se (rise in nasal wash cells, virus shedding with peak on T+59, sero-conversion to H1N1), but showed immune protection against disease in terms of reduced weight loss relative to the control group 4.

[0186] Thus, aerosol delivery of low doses of (a) formalin inactivated H1N1 virus or (b) in situ generated UV inactivated H1N1 virus three times to the lungs resulted in the generation of immune protection against future challenge with or exposure to infectious H1N1 virus demonstrated by significantly reduced clinical symptoms in particular significantly reduced weight loss.

[0187] This immune protection was seen where an HAI titre was not detectable, and therefore below that often taken as required for effective protection.

[0188] The immune protection described here is expected to apply to all airborne pathogens; it is expected to apply to all airborne viruses, all airborne RNA viruses, all airborne negative strand RNA viruses, to all orthomyxoviruses, to all influenza viruses and/or to influenza A viruses.

[0189] Based on the effective dose of 500 PFU, it can be deduced that doses of similar magnitude or larger stimulate the immune system and will also be effective if delivered by the same route and in repeated doses. It is known that repeat immunising will boost the immune responses therefore based on these observations it is recognised that for significantly lower doses than 500 PFU more than 3 immunisations are likely to be required. Clearly the 500 PFU or similar may be inactivated by UV or formalin or similar inactivating agent, and may be provided as a manufactured vaccine preparation or inactivated in situ.