Efficacy of nucleoprotein and haemagglutinin antigens expressed in fowlpox virus as vaccine for influenza in chickens

Papers Efficacy of nucleoprotein and haemagglutinin antigens expressed in fowlpox virus as vaccine for influenza in chickens Robert G. Webster *~, Yoshihiro Kawaoka*, Jill Taylor t, Randall Weinberg t and Enzo Paoletti t Fowlpox virus (FPV) recombinants expressing influenza virus H5 haemagglutinin (HA), nucleoprotein (NP) or co-expressing both of these antigens were tested for vaccine efficacy in chickens. Immunization with the recombinant FPV-HA was highly efficacious but provided no cross protection between subtypes. Bursectomy established that immunity against the H5 subtype was antibody-mediated despite the presence of very low levels of antibody in the vaccinated birds. Immunization with the recombinant F P V expressing the cross-reactive N P antigen did not provide protective immunity despite hyperimmun&ation and provided no benefit above HA expressed alone. The results suggest that the kinetics of viral replication outpaces immunity induced by NP. Keywords:Fowlpoxvirus; chickens;recombinants;influenza

INTRODUCTION Immunity to influenza is mediated by both humoral and cell-mediated responses. The viral proteins of primary importance are the haemagglutinin (HA) that induces neutralizing antibodies and the nucleoprotein (NP) that is important in cytotoxic T cell-mediated immunity t. Other influenza viral proteins, including the neuraminidase (NA) and polymerase, also induce both B- and T-celt immunity to influenza viruses, but are less important in eliciting initial protection and recovery from infection. Humoral immunity to the HA is subtype-specific, but accumulation of point mutations (antigenic drift) permits the virus to escape from immune surveillance. On the other hand, cell-mediated responses to the NP are cross-reactive between the different subtypes of influenza A viruses 2 and are not sensitive to antigenic drift. Since NP is highly conserved and cross-reactive with all influenza A viruses, a vaccine that is based on both the HA and NP may be advantageous. Studies in mice have established that HA expressed in a vaccinia recombinant induces protection from challenge with influenza virus, whereas NP does not 3'4. On the other hand, in vitro cultured and stimulated NP-specific cytotoxic T lymphocytes (CTL) can protect mice from influenza virus challenge 3. The question addressed here *St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38101-0318, USA. tVirogenetics Corporation, 465 Jordan Road, Rensselaer Technology Park, Troy, New York 12180-8349, USA. ~tTowhom correspondence should be addressed. (Received 31 May 1990; revised 14 September 1990; accepted 17 September 1990) 0264-410X/91/050303-06 /n 1991Butterworth-HeinemannLtd

is whether chickens respond to influenza virus antigens expressed in a fowlpox vector in a similar fashion to mice and whether co-expression of both the HA and NP proteins can influence the immune status. In addition, influenza is a natural infection in chickens in which the highly virulent strains of the H5 and H7 subtypes become systemic and the requirements for protection may be different from in mice. Since the HA and NP of influenza viruses are thought to be of primary importance in immunity to influenza, we have investigated the efficacy of these proteins expressed singly5 and together in a fowlpox recombinant. NP expressed alone or co-expressed with HA provided no detectable protection or cross-protection that could not be explained by HA alone. On the other hand, the HA alone induced long-lasting immunity that was abolished by bursectomy. In two of the three experiments, there was evidence for reduced levels of protection in chickens vaccinated with the fowlpox recombinant co-expressing HA and NP. MATERIALS AND METHODS Viruses The avirulent and virulent H5 and H7 influenza viruses used in this study were from the repository at St Jude Children's Research Hospital (Tables I-3), they included A/Turkey/Ireland/1378/83 (H5N8) [Ty/Ire], A/Chick/ Penn/1370/83 (H5N2) [Ck/Penn] and A/Chick/Victoria 1/85 (H7N7) [Ck/Vic]. Viruses were grown in 11-day-old embryonated chicken eggs and were purified by equilibrium sedimentation through 25-70% sucrose gradient in a

Vaccine, Vol. 9, May 1991 303

Immunity to influenza." R.G. Webster e t al. Table 1

Protection of chickens with influenza antigens expressed in a fowlpox recombinant Protection

Virus detection a

Challenge virus

Vaccine

Age of chickens

Sick/dead/total

Trachea

Cloaca

Ty/Ire/83 (H5N8)

VFP-14 (HA)

2 4 2 4 2 4 2 4 2

days weeks days weeks days weeks days weeks days

0/0/11 0/0/5 0/0/10 1/1/5 10/7/10 3/3/5 10/7/10 5/3/5 10/9/10

0/11 0/5 0/10 0/5 1/10 1/5 2/10 0/5 0/10 a

0/11 0/5 0/10 0/5 5/10 1/5 4/10 2/5 4/10 a

None

2 4 2 4 2 4 2 4 2

days weeks days weeks days weeks days weeks days

0/0/6 0/0/5 2/2/6 0/0/6 6/3/6 5/2/5 6/1/6 " 5/3/5 5/2/5

5/6 5/5 6/6 6/6 6/6 5/5 5/5 5/5 5/5

2/6 3/5 2/6 3/6 6/6 4/5 5/5 5/5 5/5

VFP-14 (HA) VFP-15 (HA + NP) VFP-12 (NP) Fowlpox control None

2 2 2 2 2

days days days days days

5/5/5 6/6/6 5/5/5 6/6/6 5/5/5

3/3 5/5 2/2 4/4 4/4

3/3 5/5 1/2 4/4 3/4

VFP-15 (HA + NP) VFP-12 (NP) Fowlpox control None Ck/Penn/1370/83 (H5N2)

VFP-14 (HA) VFP-15 (HA + NP) VFP-12 (NP) Fowlpox control

Ck/Vic/85 (H7N7)

Chickens were challenged with 109 LD,o of the highly pathogenic A/-rurkey/Ireland/1378/83 (H5N8) or A/Chick/Penn/1370/83 (H5N2) influenza viruses by administering 0.1 ml to the nares of each bird. Two-day-old birds were challenged 4 weeks after vaccination and 4-week-old birds were challenged at 5 weeks postvaccination. Swelling and cyanosis of the face and comb and haernorrhage of the legs were taken as an indication of sickness; such birds frequently could not stand. The birds were sampled 3 days after infection by tracheal and cloacal swabbing. Virus was detected by inoculation of embryonated eggs "Virus detection in tracheal and cloacal sample was low after infection of chickens with Ty/Ire/83 (H5N8) but the virus caused high mortality. This virus is more pathogenic in turkeys than in chickens

Table

2

Serological response induced by inoculation of chickens with influenza virus antigens expressed in fowlpox recombinants HI titres to: Ty/Ire/83

Challenge virus

Vaccine

Ty/Ire/83 (H5N8)

VFP-14 (HA) VFP-15 ( H A + N P ) VFP-12 (NP) Fowlpox parent None

Ck/Penn/83 (H5N2)

VFP-14 (HA) VFP-15 (NP + HA) VFP-12 (NP) Fowlpox parent None

Ck/Penn/1370/83

Age of chickens

Post-Vacc

Post-Chall

Post-Vacc

Post-Chall

2 4 2 4 2 4 2 4 2

days weeks days weeks days weeks days weeks days

10 45 5 5 < 10 <10 <10 < 10 <10

1 600 280 750 550 < 10 <10 750 (3) 400 (2) 320 (1)

< 10 16 <10 < 10 < 10 <10 <10 < 10 <10

1 300 200 400 400 < 10 <10 320 (3) 240 (2) 160 (1)

2 4 2 4 2 4 2 4 2

days weeks days weeks days weeks days weeks days

50 20 5 8 < 10 <10 <10 < 10 <10

550 1 500 600 (4) 750 < 10 <10 64 (5) 100 (2) 100 (3)

10 8 4 2 < 10 <10 <10 < 10 <10

1 750 600 280 1 400 < 10 <10 1 150 320 1 100

(4)

ELISA titres to NP: Post-Vacc

Post-Chall

<100 < 100 < 100 <100 -

3000 3 000 100 000 300000

< 100 < 100 < 100 <100

1 000 10000 300 000 300000

(5) (2) (3)

The 4-week-old birds were bled before vaccination and tested in HI and ELISA tests. None contained detectable antibody levels and the results are not shown. The 2-day-old chickens were bled at 6 weeks postvaccination (Post-Vacc) and the 4-week-old birds were bled at 5 weeks postvaccination; both groups were bled 2 weeks after challenge (Post-Chall). The figures are the mean antibody titres from the same groups of chickens described in Table 1. The numbers in parentheses are those that survived challenge < , less than 10; , not tested

Beckman SW28 rotor (Beckman, Palo Alto, CA). Virion RNA was isolated by treatment of purified virus with proteinase K and sodium dodecyl sulphate, followed by extraction with phenol:chloroform (1:1) as described previously6.

304

V a c c i n e , Vol. 9, M a y 1991

Construction of fowlpox influenza HA/NP recombinants A c D N A copy of the HA gene of the highly virulent Ty/Ire and Ck/Penn, respectively, has been cloned and sequenced v. In these studies, a c D N A copy of the N P

I m m u n i t y to influenza: R.G. Webster et al. Table 3

Influence of hyperimmunization with nucleoprotein on protection of chickens against challenge with virulent influenza virus Protection

Virus detection

ELISA titres

Challenge virus

Group

Sick/dead/total

Trachea

Cloaca

Post-Vacc

Post-Boost

Post-Chall

Ck/Penn (H5N2)

VFP-12 (NP) Control

6/2/6 3/3/4

6/6 4/4

6/6 3/4

1000

10000

100000(2)

Chickens (4-week-old) were immunized by infection with fowlpox expressing influenza A NP, 2 weeks later the birds were hyperimmunized by intraperitoneal injection of fowlpox NP grown in the chorioallantoic membrane of 11-day-old chicken embryos. Chickens were infected with Ck/Penn (H5N2) 1 week after boosting as given in Table 1

Table 4

Effect of chemical bursectomy on protection mediated by influenza antigens expressed in fowlpox Protection

Virus detection

HI antibody to Ck/Penn

HI antibody to Ty/Ire

Challenge virus

Group

Treatment

Sick/dead/total

Trachea

Cloaca

Post-Vacc

Post-Chall

Ck/Penn (H5N2)

VFP-14 (HA)

None

0/0/4

2/4

0/4

< 10

140

Cyclophosphamide None

5/4/6 2/2/9

6/6 9/9

4/6 3/9

<10 < 10

40(2) 80(7)

<10 < 10

80(2) 140(7)

Cyclophosphamide

5/5/6

6/6

6/6

< 10

320(1)

< 10

2560(1)

VFP-15 (NP+HA)

Post-Vacc 40

Post-Chall 280

Chickens were chemically bursectomized with cyclophosphamide (Cytoxan"). The chicks received 4 mg cyclophosphamide intraperitoneally each day for 3 days beginning the day after hatching. At 7 weeks of age, the birds were vaccinated by wing web puncture as given in Table 1. Four weeks after vaccination, the birds were infected with Ck/Penn (H5N2) as given in Table 1

gene of the avirulent A/Chick/Penn/I/83 (H5N2) 6 was utilized. The recombinant fowlpox vFP-12, was constructed by the insertion of a eDNA copy of the N P gene at a unique Hind II site of 5.1 kbp PvuII fragment of fowlpox virus (FPV) DNA. Transcription of the inserted gene was regulated by placement of the previously described vaccinia virus H6 promoter s'8'9 at the 5' terminus of the gene. The fowlpox recombinant vFP-14, was constructed by insertion of a eDNA copy of the H5 HA of Ty/Ire at a BamHI site of an approximately 10 kbp PvuII fragment of FPV DNA. Transcriptional regulation of this gene was also controlled by the vaccinia virus H6 promoter. The double recombinant vFP-I 5 was created in a similar manner using the N P single recombinant, vFP-12 as the recipient virus. Recombinant viruses were isolated and processed by methods previously described 1°. Immunofluorescence experiments were performed essentially as described using a monoclonal antibody pool specific for the H5 HA or a polyclonal goat anti-NP sera. For examination of intracellular antigens, cells were fixed with acetone for 5 min at 20°C prior to exposure.

and screened for virus by inoculation into embryonated eggs 6.

Vaccination and challenge of chickens

Characterization of FPV-HA/NP recombinants

Specific pathogen-free (SPF) white leghorn chickens (2 days and 5 weeks old) were vaccinated by wing web puncture with a double needle used for commercial vaccination of poultry with FPV. Approximately 2/A inoculum containing 6× 105 plaque-forming units of recombinant FPV virus was given to each bird. The older birds were bled prior to challenge and 2 weeks later. Birds were challenged with l03 LDso of virus administered in 0.1 ml to the nasal cleft. The birds were observed daily for disease signs which included swollen faces, malaise, loss of appetite, diarrhoea, cyanosis of the comb or wattles, paralysis and death. Most deaths occurred between 4 and 7 days after infection. Tracheal and cloacal swabs were taken from each chicken 3 days after infection

The H5 (Ty/Ire) HA gene inserted in recombinant vFP-14 has been shown to be expressed on the infected cell surface and correctly cleaved into HA1 and HA2 subunits 5. The recombinant vFP-12 containing the N P gene was shown by immunoprecipitation analysis to express a protein of approximately 55kDa. Although strong cytoplasmic fluorescence in infected chick embryo fibroblast (CEF) cells could be demonstrated, surface fluorescence was not conclusively shown. Western blot analysis using polyclonal sera specific for the HA or N P demonstrated that equivalent amounts of both proteins are expressed by the double recombinant vFP-15 as by the two single recombinants, vFP-12 and vFP-14 (protein expression results are not shown).

Chemical bursectomy of chickens SPF chickens were injected intraperitoneally with 4rag cyclophosphamide daily for 3 days beginning the day after hatching. Bursectomy was evaluated by the inability of the birds to produce antibodies to influenza virus (Table 4).

Serological tests HA titrations and haemagglutination-inhibition (HI) tests were performed in microtitre plates with receptordestroying enzyme-treated sera tl. Enzyme-linked immunosorbent assays (ELISA) were done as described 12 For assay of antibodies to the N P the ELISA plates were coated with A/Duck/Memphis/546/76 (HI1N9) that contains type A-specific N P antigen.

RESULTS

Vaccine, Vol. 9, M a y 1991

305

Immunity to influenza: R.G. Webster et al.

lmmunogenicity of FPV-HA, -NP, -HA/NP recombinants in chickens To assess the immunogenicity of FPV recombinants expressing either HA, N P alone or co-expressing both HA plus NP, chickens were vaccinated by inoculation with the FPV recombinants. Groups of 2-day-old and 4-week-old chickens were vaccinated with the three different fowlpox recombinant vaccines and subsequently challenged with homologous H5 (Ty/Ire), variant H5 (Ck/Penn) and heterologous H7 (Ck/Vic) influenza viruses (Table I ). All of the vaccinated chickens developed a typical pox lesion at the site of vaccination on the wing web by the third day, scabs formed by 5 days and the lesion completely resolved by 7 days. No secondary lesions formed in any of the birds and the fowlpox recombinant vaccines did not spread to non-vaccinated control birds housed in the same cages. The results of challenge experiments are shown in Table 1 and the serological results in Table 2. Chickens of either age vaccinated with FPV-HA or F P V - H A / N P were largely protected from challenge with homologous H5 and variant H5 viruses, but were completely susceptible to infection with virulent H7N7 influenza viruses. Birds immunized with F P V - N P or F P V - H A / N P provided no protection from challenge with homologous or heterologous virus that could not be accounted for by protection mediated by HA alone. In the groups vaccinated with F P V - H A / N P , one of five birds (4-week-old) died after challenge with Ty/Ire and two of six 2-day-old birds died after challenge w i t h Ck/Penn. This is surprising for none of the birds vaccinated with HA alone (Tables 1 and 4) or in earlier studies 5 showed any disease signs or died of infection, raising the possibility of lower vaccine efficacy when HA and N P are co-expressed in the FPV recombinant. The vaccines containing HA induced low levels of HI antibody to the homologous virus (Ty/Ire) after initial vaccination (range 1/5 to 1/50) (Table 2). The reactivity of these antibodies with the variant H5 (Ck/Penn) was lower than with the homologous virus (~five- to tenfold) and in some groups could not be detected. The low levels of cross-reactive antibodies did not prevent infection of the chickens after challenge with the variant H5 virus (Ck/Penn) consistent with previous data 5. Despite virus shedding, none of the birds that received the FPV-HA vaccine and were subsequently challenged with Ck/Penn showed any disease signs or died. Antibody to N P as measured by ELISA was not detectable after vaccination (Table 2), but after challenge high levels of antibody to N P were detected in the birds vaccinated with F P V - N P indicating that the birds were probably primed. Birds immunized with F P V - H A / N P had lower levels of N P antibody after challenge suggesting that the co-expressed N P was perhaps less immunogenic.

Will hyperimmunization with NP induce protection? The results from the above experiments indicated that low levels of humoral immunity were induced by F P V - N P and that these birds were not protected from challenge with homologous or heterologous influenza virus. To determine if hyperimmunization would induce higher levels of immunity, a group of chickens were given two doses of vaccine before challenge (Table 3). After initial vaccination, antibodies were detected by ELISA

306

Vaccine, Vol. 9, May 1991

(1000) and after boosting the levels increased to 10000. Despite the high levels of humoral antibody to NP, virus replication occurred in all birds and all of the birds showed severe disease signs typical of highly pathogenic influenza. Although only two of the six birds died, the surviving birds were chronically ill and the results were not different from the non-vaccinated control group.

Is cell-mediated immunity providing cross protection within the H5 subtype? Although the H5 (Ty/Ire) expressed in FPV induced very low or non-detectable levels of antibodies that cross-reacted with the variant H5 (Ck/Penn) influenza virus, the birds were protected from death and disease signs (Tables I and 2). We wondered how these birds were protected from developing disease signs, especially since virus replication occurred in all birds (Table I). It is possible that cell-mediated immunity may play a role in clearing virus before it reached critical sites, or alternatively, the antibodies that reacted with Ty/Ire served to prime the birds for an amnestic response after infection with the variant H5 virus (Ck/Penn). To distinguish between these possibilities, groups of birds were chemically bursectomized (Table 4), vaccinated with FPV-H5 (Ty/Ire) and subsequently challenged with the variant H5 virus (Ck/Penn). Since B-cell development in the chicken occurs in the bursa of Fabricius, destruction of this organ interferes with antibody production, but leaves the thymus-dependent cell-mediated response largely intact 13. If cell-mediated responses were responsible for immunity after vaccination with FPV-H5 (Ty/Ire) and challenge with Ck/Penn, then bursectomy. should not have abolished the ability of the birds to develop a protective immune response. The results show that bursectomy did abolish protective immunity (Table 4). Two of the cyclophosphamide-treated birds vaccinated with FPV-H5 that survived challenge with Ck/Penn influenza virus did develop low levels of antibody after challenge, indicating that bursectomy was incomplete. The control groups of birds (not bursectomized) showed no disease signs, but in the control group vaccinated with the F P V - H A / N P two of nine birds died.

Does simultaneous or co-expression of HA plus NP produce cross-reactive immunity? In the above experiments, a small number of birds vaccinated with the FPV construct that co-expresses HA plus N P died after challenge with the homologous H5 or variant H5 viruses (Tables ! and 4). Although not statistically significant, the results raise the possibility that co-expression may reduce the efficacy as compared with HA and N P expressed separately. Studies were done to determine if vaccination with HA and N P given singly, simultaneously or co-expressed in FPV resulted in comparable protection (Table 5). In these studies, all birds vaccinated with HA or HA plus N P were protected. As in the initial experiment, N P provided no protection and subsequent challenge with another subtype of influenza (Ck/Vic) resulted in the death of the birds that survived the H5 challenge. The mortality in the groups that received co-expressed HA and N P (Tables I and 4) was not repeated in this experiment, but does indicate that there is no benefit to be derived from the use of a vaccine that co-expresses HA plus NP. The birds in this experiment were challenged ~1 week later than in tile earlier studies.

Immunity to influenza: R.G. Webster et al. Table $

Protection of chickens with influenza antigens expressed in fowlpox administered singly or co-expressed Challenge with Ck/Penn (H5N2) Virus replication

Challenge of survivors with Ck/Vic (H7N7)

Challenge virus

Group

Sick/dead/total

Trachea

Cloaca

Sick/dead/total

Ck/Penn (H5N2)

VFP-14 (HA) VFP-12 (NP) VFP-15 (NP + HA) VFP-12 (NP) VFP-14 (HA) No vaccine control

0/0/10 10/9/10 0/0/7 0/0/12

2/10 7/10 1/7 2/12

0/10 3/10 0/7 1/12

10/10/10 1/0/1 7/7/7 12/11/12

10/10/10

5/10

8/10

Chickens (2-day.-old) were immunized in the wing web and challenged with virus as given in Table 1

DISCUSSION The concept of a vaccine for influenza that is based on cross-reactivity provided by N P with specificity provided by HA would be advantageous both in chickens and humans. In the present study, we investigated this concept in chickens with N P and HA expressed in FPV. The constructs contained HA or N P alone or HA plus N P co-expressed in FPV. Immunoprecipitation analysis demonstrated the expression of the appropriate gene products in both single and double recombinants. No difference in the expression levels of either protein was apparent by Western blot analysis. Each of the constructs produced typical pox lesions after vaccination of chickens in the wing web, no secondary lesions developed and the vaccine strains did not transmit to susceptible contact birds. The 2-day- and 5-week-old chickens vaccinated with FPV-HA were completely protected from challenge with the homologous T.y/Ire virus, no virus replication was detected and no disease signs developed. These birds were also protected from disease signs and death when challenged with a virulent variant H5 influenza virus (Ck/Penn); however, the birds were infected and local virus replication occurred. On the other hand, chickens vaccinated with F P V - N P were not protected from infection or disease signs and the majority of birds died with signs of highly pathogenic influenza. It is apparent that N P does not provide protection of chickens even after hyperimmunization. These results are similar to what has been found in mice immunized with vaccinia-NP recombinants 3'14. In the mouse system, a significant component of the CTL response to influenza virus is directed at cells bearing peptides derived from the N P 15 and memory to N P antigen persists for many years 16. Why then is the mouse or the chicken not protected from challenge with influenza after vaccination with N P expressed in recombinant pox viruses? The problem is perhaps associated with the kinetics of the cell-mediated responses and the necessity for localization of CTLs in the lungs. In vivo-stimulated NP-specific CTLs cannot efficiently move to the influenza-infected lungs, whereas in vitro-stimulated CTLs preferentially move to the lung iv. The kinetics of influenza viral replication outpaces the CTL response and movement of cells to the lungs; consequently, cell-mediated responses induced by vaccinia-NP are ineffective at providing initial protection from infection. We do not know if the same features of cell-mediated immunity are found in chickens, but the end result is the same.

CTL responses were not determined in these studies, but from the NP-specific antibody response we know that the chickens did develop an immune response to FPV-NP. The bursectomy experiments (Table 4) established that antibody-mediated immunity to the HA was responsible for protection against challenge with the variant H5 (Ck/Penn) even though the levels of antibodies were very low or undetectable. These studies provide an answer to a question that could not be resolved in the mouse influenza system. Andrew and Coupar s showed that mice 4mmunized with vaccinia HA were protected despite very low levels of antibody. They could not determine whether this was B or T cellmediated. The present studies indicate that low levels of antibody to HA can elicit protection. The co-expression of HA and N P in an FPV recombinant did not improve the efficacy of the HA vaccine in chickens. A small and statistically insignificant number of birds immunized with FPV-HA/N P developed disease signs and died after challenge (Tables I and 4). Since none of the chickens immunized with HA alone developed" disease signs or died after challenge, it is possible that N P co-expressed with HA may have been less effective than if both were expressed simultaneously. There was no evidence for improved efficacy with simultaneous or co-expression of HA and NP in FPV and the question of reduced efficacy in the F P V - H A / N P groups remains questionable. Influenza vaccines are not widely used in the poultry industry in the USA. An oil-water emulsion of allantoic fluid containing influenza is available as vaccine but is not widely used. Influenza in turkeys is a continuing problem in the USA but occurs less frequently in chickens. Regardless, devastating outbreaks have occurred in chickens in many countries in the past 10 years is. The FPV-expressed HA vaccine described here is highly efficacious, easy to administer and would be crossprotective within the subtype. This vaccine has potential utility both in chickens and turkeys. ACKNOWLEDGEMENTS This work was supported in part by Public Health Service grants AI 08831 and AI 29599 from NIAID the National Institutes of Health, Cancer Center Support (CORE) Grant CA 21765 and by American Lebanese Syrian Associated Charities. The authors are grateful to Tim Thomas and Johanna Van der Hoeven for excellent technical assistance and Dr Elizabeth Norton for

Vaccine, Vol. 9, M a y 1991

307

I m m u n i t y to i n f l u e n z a : R.G. W e b s t e r et al.

immunofluorescence analysis. Experiments using Ty/Ire, Ck/Penn, and Ck/Vic viruses were performed in BL 3 containment facilities. The authors thank Dayna Anderson for manuscript preparation. REFERENCES 1 Yewdell, J.W. and Hackett, C.J. Specificity of function of T lymphocytes induced by influenza A viruses. In: The Influenza Viruses (Eds Fraenkel Conrat, H. et al.) 1989, Plenum Press, New York, pp. 361-429 2 Zweerink, H.J., Courtneidge, S.A., Skehel, J.J., Crumpton, M.J. and Askonas, B.A. Cytotoxic T cells kill influenza virus infected cells but do not distinguish between serologically distinct type A viruses. Nature (London) 1977, 267, 35z~356 3 Andrew, M.E. and Coupar, B.E.H. Efficacy of influenza haemagglutinin and nucleoprotein as protective antigens against influenza virus infection in mice. Scand. J. Immunol. 1988, 28, 81-85 4 Doherty, P.C., Allan, W., Boyle, D.B., Coupar, B.E.H. and Andrew, M.E. Recombinant vaccinia viruses and the development of immunization strategies using influenza virus. J. Infect. Dis. 1989, 6, 1119-1121 5 Taylor, J., Weinberg, R., Kawaoka, Y., Webster, R.G. and Paoletti, E. Protective immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine 1988, 6, 504~508 6 Bean, W.J., Kawaoka, Y., Wood, J.M., Pearson, J.E. and Webster, R.G. Characterization of virulent and avirulent A/Chicken/Pennsylvania/83 influenza A viruses: Potential role of defective interfering RNAs in nature. J. Virol. 1985, 54(1), 151 160 7 Kawaoka, Y., Nestorowicz, A., Alexander, D.J. and Webster, R.G. Molecular analyses of the hemagglutinin gene of H5 influenza viruses: Origin of a virulent turkey strain. Virology 1987, 158, 218 8 Guo, P., Goebel, S., Davis, S., Perkus, M.E., Languet, B., Desmettre, P., Allen, G. and Paoletti, E, Expression in recombinant vaccinia virus of the equine herpesvirus 1 gene encoding glycoprotein gp13

308

Vaccine, Vol. 9, M a y 1991

9

10

11

12

13 14

15

16

17

18

and protection of immunized animals. J. Virol. 1989, 63, 4189-4198 Perkus, ME., Limbach, K. and Paoletti, E. Cloning and expression of foreign genes in vaccinia virus using a host range selection system. J. Virol. 1989, 6,3, 3829-3836 Panicalli, D. and Paoletti, E. Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc. Natl Acad. Sci. USA 1982, 79, 4927-4931 Palmer, D.F., Coleman, M.T., Dowdle, W.R. and Schild, G.C. Advanced laboratory techniques for influenza diagnosis. In: Immunology Series No. 6 US Department of Health, Education, and Welfare, 1975, pp. 51-52 Kida, H., Brown, L.E. and Webster, R.G. Biological activity of monoclonal antibodies to operationally defined antigenic regions on the hemagglutinin molecule of A/Seal/Massachusetts/I/80 (H7N7) influenza virus. Virology 1982, 122, 38-47 Glick, B., Chang, T.S. and Jaap, R.G. The bursa of Fabricius and antibody production of the domestic fowl. Poult. Sci. 1956, 35, 224 Stitz, L., Schmitz, C., Binder, D.~ Zinkernagel, R., Paoletti, E. and Becht, H. Characterization and immunological properties of influenza A nucleoprotein. Cell associated NP isolated from infected cells or viral NP expressed by vaccinia recombinant virus do not confer protection. J. Gen. Virol. 1990, 71, 1169-1179 Townsend, A.R.M., Rothbard, J., Gotch, F.M., Bahadur, G., Wraith, D. and McMichael, A.J. The epitopes of influenza nucleoprotein recognized by'cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 1986, 44, 959-968 Effros, R.B. and Walford, R.L. The effect of age on the antigenpresenting mechanism in limiting dilution precursor cell frequency analysis. Cell Immunol. 1984, 88, 531-539 McDermott, M.R., Lukacher, A.E., Braciale, V.L., Braciale, T.J. and Bienenstock, J. Characterization and in vivo distribution of influenzavirus-specific T lymphocytes in the murine respiratory tract. Am. Rev. Respir Dis. 1987, 135, 245 Webster, R.G. and Kawaoka, Y. Avian influenza. In: Critical Reviews in Poultry Biology, 1988, CRC Press Inc. Boca Raton, Florida, pp. 211 246