HA2 influenza virus protein

Influenza A subtype cross-protection after immunization of outbred mice with a purified chimeric NS]/HAz influenza virus protein Innocent N. Mbawuike *~, Susan B. Dillon t, Sandra G. Demuth t, Christopher S. Jones ~, Thomas R. Cate* and Robert B. Couch* Influenza A/PR/8/34-derived chimeric (D) protein (SK&F 106160) composed of the first 81 amino acids (aa) of NSI fused to the conserved 157 C-terminal aa of HA2 (NSII-81-HA26~-222) was previously shown to induce H-2d-restricted protective cvtotoxic T-lymphocyte ( CTL ) hnmunity in inbred mice. However, D protein, like other small peptides, exhibited haplotype dependence and was not immunogenic in H-2 b and H-2 K mice. A potential use of this antigen in humans and the role of T cells in any protection were evaluated in outbred Swiss and hTbred CBF6F/ (H-2 d/b) mice. Mice immunized with D protein and challenged by small-particle aerosol with a lethal dose of influenza virus were significantly protected against mortality from influenza A/H1N1 and A/H2N2 (p
The MHC class I-restricted anti-influenza virus cycotoxic T-lymphocyte (CTL) response plays an important role in humans 1 and mice 2-5 in limiting the spread of viral infection and promoting viral clearance. The majority of the CTL response appears to be directed to the internal nucleoprotein (NP) of the virus, resulting in a cross-reactive immune response against different influenza A subtypes 6-9. Inactivated influenza whole-virus vaccines or virus subunits, unlike infectious virus, have been shown in numerous studies to be ineffective in stimulating CTL activity, but instead induce protective antibody directed towards the haemagglutinin (HA) and neuraminidase *Acute Viral Respiratory Disease Unit, Influenza Research Center, Department of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498, USA. ?Department of Molecular Virology and Host Defense, and tDepartment of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA. =To whom all correspondence should be addressed. (Received 4 October 1993; revised 29 March 1994; accepted 30 March 1994) 0264,-410X/94/14/1340-09 1994 Butterworth-Heinemann Ltd

1340 Vaccine 1994 Volume 12 Number 14

(NA) that are restricted in effectiveness to the vaccine virus and related variants 1°-13. Antigenic variation at the antibody-binding sites on the HA1 subunit of influenza virus HA is thought to decrease the protective efficacy of these vaccines ~4.~5. The HA 2 subunit of the HA molecule is highly conserved within A/H1NI strains (--~97%), and has less, albeit marked, homology to the A/H2N2 (79%) and A/H3N2 (50%) subtypes ~6. Also, NS t is highly conserved as indicated by significant antigenic crossreactivities for NSt of all influenza A viruses tested ~7. Using synthetic peptides, recombinant proteins or viral vectors expressing influenza HA, several groups have demonstrated induction of and recognition by CTL directed to the HA t and HA 2 subunits of influenza HA 1a-23. In addition, the surface protein, NA, internal proteins (nucleoprotein, matrix and polymerase proteins) and the non-structural protein 1 (NS1) are all recognized by murine CD4 + and/or CD8 + T cells ~. Various approaches are being used to develop improved influenza vaccines, including strategies to enhance IgG responses and/or to induce local IgA responses. We have chosen an alternative approach, that

Influenza A cross-protection with a chimeric protein: I.N. Mbawuike et al.

ofeliciting cross-reactive T-cell responses by recombinant influenza antigens. In contrast to antibodies, which are specific for homologous or closely related virus strains, T cells recognize more conserved regions of viral proteins, and therefore are more cross-reactive for serologicaily distinct viruses 7. Employing prototype A/H1N1 (strain A/PR/8/34)derived recombinant proteins produced in Escherichia coli, we initially selected candidate vaccine antigens based on relative expression levels and ability to stimulate a CTL response in mice. The best success was achieved with a series of fusion proteins constructed from the HA 2 subunit of surface HA protein fused to the first 81 amino acids of the NS1 protein 24. The fusion dramatically increased expression levels of the HA2-derived proteins to levels which represented up to 20% of the total cell protein (unpublished results). These chimeric proteins were therefore chosen for further evaluation. Recently, one of the influenza A/PR/8/34-derived chimeric proteins (c13), composed of the first 81 amino acids (aa) of NSI fused to the entire 222 aa of HA 2 (NSI I-sI-HA21 222) and its derivative (D protein, N S 1 I_aI-HA265_222) were shown to induce H-2 drestricted MHC class I influenza A cross-reactive CTL in inbred mice 24. Despite an absence of neutralizing antibody, active immunization with partially purified cl 3 or D protein or adoptive transfer of CTL clones induced by them led to protection against intranasal challenge with influenza A/H1N1 and A/H2N2 but not A/H3N2 viruses 25'26. Extensive purification of D protein, however, resulted in loss of activity that was restored only when immunized in CFA emulsions or adsorbed to aluminium hydroxide adjuvant 26. As shown previously for small peptide vacines, D and c13 proteins exhibited haplotype dependence, as they were not reactive in H-2 b and H-2 K mice 25-27. Since it has been suggested that haplotype restriction could potentially limit the usefulness of peptide vaccines in humans 28, there is a need to evaluate such candidate vaccines in outbred species prior to study in humans. Even though other outbred animal models (ferrets and squirrel monkeys) have been used to study influenza disease 29, the mouse is a more suitable model because of the availability of well characterized immunoreagents and the ability to evaluate T-cell responses in inbred strains. Also, historically, immune responses to influenza infection in mice have always predicted responses in humans ~'29. In a series of studies designed to compare antibody responses to commercial influenza vaccines and virus infections, good correlations between human ancI animal (mice, hamsters and squirrel monkeys) responses were demonstrated 3°-32. The present study was initiated to evaluate the protective efficacy of D protein in outbred mice to represent more closely evaluation in outbred human populations. The role of T-cell subsets (CD4+ and CD8 +) in in uivo protection against virus challenge was determined. The small-particle aerosol (SPA) system for influenza virus challenge was utilized to approximate better the primary mode of natural infection. MATERIALS AND METHODS Mice Age-matched 4--8-week-old female outbred NIH Swiss and Balb/c (H-2 d) ~ x C57BL/6 ~ (H-2 b) hybrid (H-2 d/b) mice (CB6FI) were purchased from Harlan Sprague

Dawley, Indianapolis, IN, and Charles River Laboratories, Wilmington, MA, respectively. All mice were certified specific pathogen-free and were devoid of adventitious virus. They were housed under a 12 h light/dark cycle in barrier filter microisolation cages and given food and water ad libitum. Viruses Challenge pools of mouse-adapted influenza viruses, A/Puerto Rico/8/34 (A/PR/8/34, H 1N 1), A/I-long Kong/68 (A/HK/68, H3N2), A/Taiwan/1/86 (H 1N 1) and B/Lee/40 were prepared by serially passaging each virus in mice as described previously 33. A mouse 50% lethal dose (LDso) following adminstration by SPA was determined for each virus as described previously 33'34. For CTL and serological studies, influenza A/PR/8/34, A/Taiwan/l/86 and B/USSR/I/86 viruses were propagated in the allantoic cavity of 10-day-old embryonated eggs. After 3 or 4 days, the allantoic fluid was harvested, clarified by centrifugation at 1800g and stored in aliquots at -70°C. The 50% tissue-culture infectious dose (TCIDso) was determined by growth in Madin-Darby canine kidney (MDCK) cells, as previously described 33'35. Recombinant D protein Influenza D protein (SK&F 106160) was constructed from influenza A/PR/8/34 (H1N1)-derived eDNA to produce a chimeric protein consisting of the first 81 amino acids (aa) of the non-structural protein (NS1) fused via a three aa linker (glutamine-isoleucine-proline) to the carboxy terminal 65-222 aa of the haemagglutinin HA 2 subunit 24"25'27. The hybrid protein (NS1 1_81-HA2 65-222)" was then expressed in E. coli using the pASI vector system 24"25'27. A highly purified D protein suitable for human vaccine use was prepared by sequential chromatography under reducing conditions on DEAE, Superose-12 and G-25 columns. The purified product migrated as a single 28 kDa band on SDS-PAGE gels, and reacted in Western blots with rabbit antisera to A/PR/8/34 virus and murine monoclonai antibody (mAb) specific for NSrt 26 Immunization of mice and SPA virus challenge Mice were given one or two subcutaneous (s.c.) injections of D protein (5-100/~g) diluted in 5% dextrose containing 200/~g alum (Rehsorptar; Armour Pharmaceuticals, Kankakee, IL), an aluminium hydroxide gel that contains 2% w/v A120 3 (10.6 mg ml- 1 AI3 +); 3 weeks later they were given an intraperitoneal (i.p.) injection with D protein alone. Another set of mice were immunized sequentially at 3-week intervals by an s.c. injection of D protein mixed at a 1:1 (v/v) ratio with Freund's complete adjuvant (CFA), followed by s.c. injection of D protein mixed with incomplete Freunti's adjuvant (IFA) and finally by i.p. injection of D protein alone. Positive control mice were immunized with non-lethal doses (0.05 LDso) or A/PR/8/34 influenza virus by SPA 33'36. Immunized mice were challenged by SPA 33'34" 3-6 weeks after final vaccinations with 3 LDso of virus and observed for 18-21 days for mortality. Protection was expressed as percentage reduction in mortality rates for the experimental group compared with the control group.

Vaccine 1994 Volume 12 Number 14

1341

Influenza A cross-protection with a chimeric protein: I.N. Mbawuike et al.

Quantification of lung virus Lungs from infected mice were obtained aseptically at different times following virus challenge and homogenized in vials containing Ipm glass beads using a Minibeadbeater (Biospec Products, Bartlesville, OK) as previously described 34. Virus was quantified by growth in MDCK cells 3"~. Briefly, 0.5 log~o dilutions of lung specimens starting at 1:5 or 1:3 were made in minimum essential medium (MEM) and 0.2 ml each was inoculated in duplicate into 96-well tissue-culture plates containing a monolayer of MDCK cells. After incubation at 37°C overnight, the medium was removed and replaced with serum-free MEM containing 2pg m1-1 trypsin (Worthington Biochemicals Corporation, Freehold, N J). After a further incubation for 4 days at 37°C, 0.05 ml of 0.5% chicken erythrocytes was added to each well. Virus titres were expressed as the reciprocal of the last dilution exhibiting haemagglutination33'35.

Neutralizing antibody (NtAb) tests Influenza virus-specific neutralizing antibody (NtAb) was determined by a modification34"35 of the microneutralization test of Frank et al. 37. Briefly, MDCK cells were allowed to adhere in microtitre wells at 37°C for 4-6 h before aspiration of medium. Serial dilutions of sera in 0.05 ml serum-free MEM containing 2pg mltrypsin (Worthington Biochemicals) were incubated with 50-100 TCIDso of influenza virus at room temperature for 1 h. The serum-virus mixture was then transferred to the MDCK monolayer plates and incubated at 37°C for 5 days. The level of NtAb was determined by the inhibition of virus-induced haemagglutination of chicken red blood cells.

T-cell subset depletions CD4+ T cells were depleted in eivo by i.p. administration of 50-200 itg of purified rat anti-murine CD4 (lgG2b, clone GK 1.5) mAb. CD8+ T cells were depleted with either 50-300 pg of purified rat anti-murine CD8 mAb (IgG2a, clone 53.6.72) or rat hybridoma (anti-murine CD8, lgM, clone 3.155) cell-culture supernatant. The hybridoma cell lines were obtained from the American Type Culture Collection (Rockville, MD), grown in serum-free Hybridoma-SFM medium (Gibco/ BRL, Gaithersburg, MD) and antibody-purified by protein G agarose affinity chromatography. Control mice were either untreated or infused with PBS or 200 pg of purified rat IgG (Sigma Chemical Company, St Louis, MO). Each mouse was injected with 0.1-0.5ml of antibody according to the following schedule: days - 3 , - 2 and - 1 before, and days +2 and +3 after virus challenge (day 0). Efficiency of depletion was determined by flow cytometric analysis (FACS) as described below.

(FITC)-conjugated mAbs to appropriate cell-surface antigens were added and incubated for 30min. After washing with PBS-0.1% NAN3, the stained cells were fixed in 1% paraformaldehyde (Sigma Chemical Co, St Louis, MO) in PBS and analysed within 24 h on a Coulter EPICS Profile Analyzer (Coulter Corporation, Hialeah, FL).

Generation of secondary CTL response Influenza A virus-specific CTL was generated in six-well plates (Costar, Cambridge, MA) in medium consisting of RPMI 1640 supplemented with 2ram L-glutamine, 100 U ml-i penicillin, 100 pg ml-1 streptomycin (JRH Biosciences, Lenexa, KS), 5x 10-sM 2-mercaptoethanol (2-ME), 10mM HEPES buffer and 10% heat-inactivated FBS. Stimulator cells (S) were obtained by incubating 4× 106 spleen cells with A/PR/8/34 influenza virus at 37'~C for 2 h. After two washes, virus-infected stimulator cells were incubated with 20 × 106 responder cells (R) at an S:R ratio of I: 10 in 10 ml medium. After 6 days, effector cells were assayed for cytotoxicity against virus-infected 33 or D proteincoated target cells.

SJCr release assay for CTL Effector cells in 0.1 ml aliquots in quadruplicate assays were serially diluted in 96-well round-bottom microtitre plates (Coming, Corning, NY) to give the designated effector-to-target (E:T) ratios. Target cells consisted of P815 mastocytoma (H-2 d) cells infected with influenza virus and incubated at 37°C for 2 h in the presence of ICr (Na2CrO 4, specific activity 400-1200 Ci U mmol- 1 New England Nuclear, Boston, MA). Alternatively, P815 cells were incubated with D protein (50pgm1-1) for 90-120 min in the presence of 5'Cr. After one wash, cells were incubated for an additional 4 h in assay medium (RPMI 1640 supplemented as above and with 5% FBS and l mM MEM and non-essential amino acids) and washed twice with medium. Virus-infected or D protein-coated target cells (5 x 103) were then added to the effector cells at varying E:T ratios and centrifuged at 1009 for 3 rain. Plates were incubated at 37°C in 5% CO2 for 4 h, centrifuged at 300g for 10-15 min and supernatant was harvested from each well using the Skatron supernatant harvesting system (Skatron, Sterling, VA). Radioactivity (counts min- 1) was determined in a gamma counter. The percentage 51Cr release was calculated from the following formula: (experimental release - spontaneous release) (maximal release - spontaneous release)

x 100

Spontaneous release (range 15-25%) was obtained from target cells incubated with medium alone and maximal release was obtained by incubating target cells with 1% Triton X-100.

Flow cytometry Immunofluorescent staining of cells and FACS 38 were performed according to the manufacturer's specifications. Briefly, splenic cells (1 x 106), unseparated or depleted of CD4+ or CD8+ T cells, were washed with RPMI 1640 medium containing 1% fetal bovine serum (FBS). They were resuspended in phosphate-buffered saline (PBS) containing 0.1% sodium azide (NAN3) at 4°C. Fluorescein

1342 Vaccine 1994 Volume 12 Number 14

Statistics Comparison of percentage mortality among vaccine groups was performed using Fisher's Exact Z2 test. Virus and NtAb titres were compared using ANOVA. All statistical analyses were performed using True Epistat, a computer program written by Dr Tracy L. Gustapon (Richardson, TX).

Influenza A cross-protection with a chimeric protein: I.N. Mbawuike et al. 10o

RESULTS

90

Protective efficacy of D protein immunization in outbred mice and effect of alum and CFA

80

Alum

70

To confirm previous results obtained in inbred mouse models using intranasal (i.n.) virus challenge 2s'-'6, outbred NIH Swiss mice were immunized with 10 or 100/~g D protein with or without alum or CFA. Three weeks later, they were challenged by SPA with 3 LDso of A/PR/8/34 virus and observed for mortality for 21 days. As shown in Figure 1, 10 or 100yg D protein induced protection against challenge with homologous A/PR/8/34 virus. Typically, unimmunized mice started to die between 7 and 8 days after challenge and 90% of the mortality occurred by day 14. By day 21, 83 and 90% of control mice injected with alum and CFA died, respectively, while only 41% of mice immunized with I0 or 1001tg D protein (without adjuvant) died (51% protection rate, p < 0.001). Adsorption of vaccine to alum had no effect on protection after 101tg D protein, but protection after 100/~g was significantly enhanced (67% protection rate, p < 0.0001 ). Emulsification of 10 or 100 l~g D protein in CFA resulted in significantly lower mortality rates when compared with mice immunized with D protein alone (71-73% protection rates, p<0.00001). Control mice immunized with a sublethal injection of A/PR/8/34 virus were also significantly protected from homologous virus challenge.

To assess the virus specificity of protection, Swiss mice were immunized with alum-adsorbed D protein and challenged 3 weeks later with 3 LDso of A/PR/8/34 or B/Lee/40 virus. As shown in Figure 2. D protein (5 and 50yg+alum) induced dose-related protection against challenge with A/PR/8/34 virus (p < 0.001 and p < 0.00001, respectively, versus alum). In contrast, mice immunized with 50/.~g D protein + alum were not protected against B/Lee/40 virus challenge. As expected, control mice immunized with live virus were significantly protected against A/PR/8/34 challenge (p<0.000001) but not against B/Lee virus challenge. D10ug lOO

D 100 ug

90

I

7O

50

o"

40

Alum Ol0ug +e}um

6O o E

O 100 ug +alum

I

CFA

30 20

D10ug +CFA

10

D 100 ug +CFA

0

E ;,'~

60

O5ug + alum

50

r----] D5Oug + alum Live virus

40

3O 2O 10 0 A/PR/8/34

B/Leel40 Challenge virus

Figure 2 Lack of protection against influenza B virus, Mice were immunized (as in Figure 1) with 5 or 5 0 y g D protein plus alum or with live virus. They were challenged with 3 L D ~ A/PR/8/34 or BILeel40 virus by SPA. Data for 20-60 mice per group in two to four combined experiments are presented

100 90 80

70 ->" E ,;~

6O

Alum

50

D50ug + alum

40 30

Influenza B virus challenge

80

->" ~o

Live virus

Figure 1 Protective efficacy of D protein in outbred mice and effect of alum and CFA adjuvants. NIH Swiss mice were given an s.c. injection of 10 or 100yg of D protein with or without alum or CFA, followed 3 weeks later by an i.p. boost with D protein alone. Control mice received alum, CFA or live virus (0.05 LDso A/PR/8/34 by SPA). Three weeks thereafter, mice were challenged with 3 LD.5o of homologous A/PR/8/34 virus by SPA. The percentage cumulative mortality (26-29 mice per group) in two combined experiments is presented

20 10

AIPRI8134 AITatwan186AIJap1305157 A/HK/68 Challenge virus Fig'Jre 3 Influenza A subtype cross-protection. D protein-immunized mice (as in Figure 1) were challenged with 3 LD~o of AJPR/8/34, A/raiwan/1/86, A/Jap/305/57 or A/HK/68 virus by SPA, Cumulative mortality rates for two combined experiments (25-32 mice per group) are presented

Influenza A subtype cross-protection Outbred mice were immunized with 50~g D protein+alum and challenged with 3 LDso of homologous A/PR/8/34 (H 1N 1) or viruses belonging to other influenza A subtypes such as A/Taiwan/1/86 (H1N1), A/Jap/305/57 (H2N2) and A/HK/68 (H3N2). D proteinimmunized mice were significantly protected against lethal challenge with each of the first three viruses (p < 0.005-0.000001) but not against A/HK/68 virus (Figure 3). Interestingly, D protein was more efficacious against A/Taiwan and A/Jap/305/57 viruses than homologous A/PR/8/34 virus. Deaths started to occur in unimmunized mice at days 7-8 postchallenge. Except for A/PR/8/34 virus-challenged control mice, all deaths occurred by day 14 following virus challenge. Approximately 10% of the deaths occurred in A/PR/8/34-infected mice after day 14. These results suggest that the latent survival time, i.e. the time between virus challenge and occurrence of initial deaths, was similar among the four viruses. Duration of protective immunity In the experiments described above, D proteinimmunized mice were challenged I-3 weeks after the last

Vaccine 1994 Volume 12 Number 14

1343

Influenza A cross-protection with a chimeric protein: I.N. Mbawuike et al. 100

~ths

90 80 70 ->"

6o

gE

so

N

40

Alum

O 100 ug + alum

Live virus

30 20 10

AIPRISI34

A/Talwan/l/86

AITmwan/1186

Challenge virus

Duration of D protein protective immunity. D protein- or live virus-immunized mice were challenged with 3 LDso of AIPRI8/34 or A/Taiwan virus, 4 or 7 months after immunization. Data for 22-32 mice per group are presented Figure 4

antigen injection. To assess the duration of D protein-induced protection, mice were immunized with 100/ag D protein+alum or live A/PR/8/34 (0.05 LD5o) virus and challenged 4 and 7 months after the last injection with 3 LD5o of A/PR/8/34 or A/Taiwan/l/86 virus. D-protein immunization accorded a low (40%) but significant (p<0.01) protection against challenge with A/PR/8/34 virus after 4 months (Figure 4). D protein also stimulated significant protection against A/Taiwan virus challenge at 4 months (89% protection rate, p<0.005) and 7 months (76% protection rate, p < 0.0005) after the last injection. The protective efficacy of D protein against A/Taiwan at 4 months was similar to that stimulated by live A/PR/8/34 virus immunization (90% protection rate, p<0.0005). These results indicate that D-protein immunization is capable of stimulating relatively long-lasting protective immunity.

Lung virus replication and NtAb Lung virus levels were measured in infected mice 3, 6 and 9 days after infection. Lung virus levels were high on day 3, started to decline by day 6 and declined to undetectable levels by day 9 (Table 1). On day 3, the levels of lung virus were only slightly lower in D proteinimmunized mice when compared with control alum and CFA groups. Ths was to be expected since CTLs do not prevent virus infection per se 2-5'33-35. However, by day 6, virus titres were significantly lower (up to 15-fold reduction) in most D protein-immunized groups, suggesting an accelerated clearance of infection. Control mice immunized with live virus were completely protected against infection and no virus was detected in their lungs. One day before virus challenge, serum was tested for neutralizing activity against A/PR/8/34 virus; NtAb activity was not detected in serum obtained from D protein-immunized mice (Table 1). ELISA antibody to the D protein was nonetheless induced (data not shown). In contrast, immunization with live virus resulted in significant serum NtAb levels. Passive transfer of D-immune serum was not protective, while serum from mice immunized with killed virus conferred significant protection (Ref. 26, data not shown).

Effect of in vivo T-cell depletions on in vitro CTL activity To assess the level of depletion of functional CTL in rive by mAb treatment, splenic lymphocytes from D

1344

V a c c i n e 1994 V o l u m e 12 N u m b e r 14

protein-immunized CB6FI mice were stimulated in vitro with A/PR/8/34 virus for 7 days. Cytolytic activity against A/PR/8/34-infected or D protein-coated P815 target cells was measured in a 4 h 51Cr release assay. Significant lysis of both A/PR/8/34-infected (Figure5a) and D proteincoated (Figure5b) P815 target cells was exhibited by lymphocytes from untreated D-immune mice. In rive treatment with mAbs to both CD8 and CD4 antigens almost completely abrogated CTL activity against both kinds of target cells. Spleen cells obtained from CB6F] mice immunized by A/PR/8/34 virus infection exhibited similar activity against A/PR/8/34-infected P815 target cells (Figure 5c) as did D protein-immune mice; in rive depletion of CD8 + and CD4 + T cells also resulted in loss of in vitro CTL activity. Control B/USSR-infected P815 target cells were not lysed by A/PR/8/34-immune lymphocytes (Figure5d) nor by D protein-immune lymphocytes (data not shown). Mediation of protective immunity by T cells CB6F 1 hybrid and NIH Swiss outbred mice immunized with D protein were depleted of C D 8 + (Lyt2 + ) and CD4 + (L3T4) T cells in rive by i.p. injection of purified specific mAbs, as described in Materials and methods. From 3-6 days after infusion of mAb, two mice from each group were killed and the splenic lymphocytes were analysed by FACS for effectiveness of depletion. The rest of the mice were then challenged with 3-5 LDso of A/PR/8/34 virus. As shown in Table 2, infusion of purified rat mAbs to murine CD8 + (53-6-72) and C D 4 + (GK1.5) T cells resulted in 88-92% reduction of these T-cell subsets by in NIH Swiss mice. Administration of mAbs to C D 8 + or C D 4 + T cells in CB6F 1 mice (Figure6) led to decreased protection Table 1 Effect of D-protein immunization on serum NtAb titre and lung virus infection

Lung virus titre e (GMT Ioglo-F s.d.) Vaccine group

Day 3

NtAb titre

Day 6

Day 9

(GMT log 2 _+s.d.) ~

Alum

7.9±0.8

6.3_+1.3

<1.0±0

<3.0±0

D 10#g D100~g D1011g+alum D100pg+alum

7.3_+1.1 7.2-+1.1 7.5-+1.2 7.4_+1.2

5.6_+1.9c 5.6_+0.9c 6.2±1.2 5.5±1.3 c

<1.0_+0 <1.0_+0 <1.0±0 <1.0_+0

<3.0-+0 <3.0_+0 <3.0_+0 <3.0_+0

CFA D 10/tg+CFA D 100 l~g + CFA

7.9-1-0.6 6.5_+1.0 7.4-t-1.4 6.1-+1.4 7.4_+1.1 5.0-t-0.8 c

<1.0_+0 <1.0_+0 <1.0_+0

<3.0_+0 <3.0_+0 <3.0_+0

Infection"

<1.0+0 °

2.4±2.1 e

<1.0+0

12.3_+0.71

GMT, geometric mean titre "Lungs from immunized mice were obtained at indicated times after challenge with 3 LDso of A/PR/8/34 virus by SPA, homogenized, and tested for virus on MDCK cells~'~; results for six mice per group are presented. The starting dilution was 1:30; a value of 1:10 was assigned to samples with no detectable virus ~Serum was obtained by orbital bleeding 1 day before virus challenge, heat-inactivated and tested for neutralizing activity against a 100 TCIDso of A/PRI81342~'2e.The starting dilution of serum was 1:16; a value of 1:8 was assigned to samples with no detectable antibody cSignificantly different from CFA and alum groups (p <0.05) aMice were infected previously with 0.05 LDsoof NPRI8134virus by SPA eSignificantly different from all other groups (p <0.01) by Student's t test in ANOVA INtAb titre is significantly higher than each of the vaccine groups (p<0.001)

Influenza A cross-protection with a c h i m e r i c protein: I.N. M b a w u i k e et al. 30

30

a

tj

m ~o

24

o

18

b

Q

O

24

,,, co n

O. "1o

"O Q

=o

18

?

c lID

~

o

12

< o

s J

6

g

01

O

12.5:1

25:1

50:1

v

12.5:1

25:1

Effector:largat ratio 50

65 t/)

¢D

o

I1: (.)

Lo '1o o o

u

40

52

d

o~ (1. "1o Q

30

39 t.-;. nU)

t"T

~

50:1

Effector:target ratio

20

¢n

26

<

al

o

o

13

~ < - - - - e ,

0 12.5:1

10

g

-~

.J

' 25:1

~ 0 12.5:1

50:1

,

,

=

.

=.. =r- = • ,~-"~" ~ -25:1

Effector:lerget ratio

50:1

Effsctor:larget ratio

Figure 5 In vivo depletion of CD8+ and CD4+ T cells abrogates m vitro CTL activity. Splenic lymphocytes were obtained from A/PPJ8134virus- or D protein-immunized T cell-depleted CB6F 1 mice (as noted on the figure), and stimulated with A/PR/8/34 virus for 7 days. Percentage lysis by D protein-immune cells of (a) AIPRI8134-infected and (b) D protein-coated P815 target cells, and lysis by A/PR/8/34-immunized cells of (c) A/PR/8/34- and (d) B/USSR-infected P815 target cells are presented for four mice per group. A , No treatment; 0 , anti-CD8 mAb; BI, anti-CD4 mAb

100

Table 2

Depletion of T-cell subsets in vivo by mAbs"

CD4+ cells -(%)

so

Mouse no.

Treatment

CD8+ cells (%)

80

1

PBS

23.4

5.9

~" ¢u

60

2

PBS

26.6

5.5

50

D 50 u g + a l u m +anti-CD8

3 4

Anti-Lyt2 Anti-Lyt2

27.1 17.2

0.6 0.4

3 E ~

4o 30

D 50 u g + a l u m +anti-CD4

1 2

None None

28.6 30.6

5.6 8.7

20 lo

3 4

Anti-L3T4 Anti-L3T4

4.3 0.1

5.8 8.7

0

5 6

Anti-Lyt2 Anti-Lyt2

29.0 26.5

0.7 1.3

Rgure 6

i

Alum

70 D 50 u g + a l u r n

Expt 1

~'

Expt 2

aSpleen cells were obtained from D protein-immunized NIH Swiss mice undepleted (PBS, none) or depleted of C D 4 + or C D 8 + T cells, by infusion of indicated mAb. Fresh cells were stained with FITC-conjugated mAb and the frequency of antigen-expressing cells was determined using the Coulter EPICS Profile Analyzer

CBSlFJ

NIH S w i s s Mice

Depletion of protective activity by administration of mAb to CD8+ and CD4+ T cells. D protein-immunized CB6F1 and Swiss mice were depleted of CD8+ and C D 4 + T cells by i.p. infusion of cell-culture supernatants or purified 53.6.72 and GKI.5 mAbs0 respectively, as described in Materials and methods. They were then challenged with 3-5 LDso A/PR/8/34 virus by SPA. Cumulative mortality rates of one experiment (10 mice per group) for CB6F1 and two combined experiments (13-35 mice per group) for Swiss mice are presented

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(49% reduction) against A/PR/8/34 virus challenge (p<0.06). In a total of nine separate experiments, administration of mAbs to CD8+ or CD4+ in NIH Swiss mice also led to reduced protection in D proteinimmunized mice (data not shown). Figure 6 shows data from two combined experiments for which the FACS profile is available (Table2). Anti-CD4 antibody treatment reduced D protein-mediated protection from 76 to 10%, a reduction of 87% (p<0.0004) while anti-CD8+ antibody treatment reduced the protection rate to 31%, a reduction of 59% (p<0.002). Injection of control PBS or purified rat immunoglobulins had no effect on protection (data not shown). These results suggest that protection was mediated by CD8 + and/or CD4+ T cells. DISCUSSION In this study, D protein, a chimeric NS1/HA2 influenza A vaccine, elicited significant protective efficacy in outbred Swiss and inbred CB6F 1 mice against virus aerosol challenge despite previous studies 24 z7 showing D protein to be haplotype-dependent in induction of a protective immune response. This result suggests that D protein could also stimulate protective T cell-mediated immunity in outbred human populations. Lung virus infection in D-immune mice was cleared faster than in control mice without detectable serum neutralizing antibody activity, an indication of a cell-mediated immune mechanism 2-L39'4°. Similar results have been obtained by others with purified influenza nucleoprotein41 and recently by us using baculovirus-expressed NP (unpublished results). Lack of protection by adoptive transfer of D-immune serum rules out a role for antibody-dependent cellular cytotoxicity or complementmediated lysis z6. Finally, depletion of T-cell subsets in vitro 24-z7 and in vivo (Figures 5 and 6) provides evidence that CD8+ T cells were responsible for clearing virus. CD4+ T cells were required for CD8 + T-cell function v, and could also have played an effector role in the clearance of virus 7"42. Unlike killed virus vaccines, D protein provided long-lasting cross-reactive protective immunity (7 months), similar to live virus infection ~°-13.4-3. D-protein immunization resulted in significant protection against homologous A/PR/8/34 (H1N1) virus, another A/H 1N1 virus in current circulation (A/Taiwan/1/86) and A/Jap/305/57 (H2N2) that circulated 36 years ago, but not against A/HK/68 (H3N2) or B/Lee/40, in agreement with earlier results 26"27. The protective determinant on HA2 for A/H3N2 may lie outside the 157 C-terminal aa, hence the lack of pvbtection. It is not clear why there is an apparently higher level of protective efficacy against heterologc/us A/Taiwan and A/Jap/305/57 viruses than homologous/A/PR/8/34. This may be explained by differences in v~rulence among the different viruses. However, this is.~anlikelysince deaths commenced in each virus challenge, group at approximately similar times (7-8 days postchallenge), and thus the latent survival times were similar. Moreover, control mice challenged with A/PR/8/34 virus died at between 7 and 21 days, while those challenged with other viruses died at between 7 and 14 days. The present results in outbred mice confirm previous reports in inbred mice in which D protein and related proteins stimulated T-cell immunity without detectable NtAb activity24-27. It is shown here that D-protein

1346 Vaccine 1994 Volume 12 Number 14

immunization did not prevent virus infection as indicated by similar levels of lung virus among control and immunized mice 3 days after virus challenge. However, after 6 days, lung virus titres were reduced (up to 15-fold reduction) in immunized mice. This suggests that D protein induced T cells that accelerated clearance of lung virus infection. In the previous studies, D protein stimulated lymphoproliferative responses and D proteinspecific ELISA antibody 24 27. Thus, despite exhibiting allotype selection with respect to certain inbred strains of mice, D protein, in the present study, stimulated significant protective CTL immunity in outbred mice. It should be noted, however, that the level of protection in outbred mice was somewhat lower than that obtained in inbred mice, leading to a suspicion that D protein might be a suboptimal vaccine in an outbred human population. This is unlikely, however, since humans are heterogeneous at the MHC class I and II loci and the protein could interact to some degree with several allelic forms of the MHC molecules 28. In a recent human trial, D-protein immunization was found to reduce viral shedding and clinical illness after an experimental influenza A/HINI challenge in young adults, without NtAb or haemagglutination inhibition (HI) antibody responses 3s, as predicted by the present results. The role of CTL in protection in those studies was not clear, however, because influenza virus-specific CTL activity was not uniformly induced in vaccinees 44. Nonetheless, those results are consistent with these obtained in outbred mice and further evaluation of the role of CTLs in humans is warranted. In the present study, in vivo depletion of either CD4 + or CD8+ T cells resulted in abrogation of in vitro-inducible CTL and suggests that both CD4 and CD8 T cells are required for protection. However, in vitro depletion and blocking experiments ~° showed that CD8 + T effector cells were responsible for in vitro lysis of virus-infected cells. Our inability to completely abrogate protection by treatment with mAb to CD8 may be due to incomplete depletion of CD8 T cells. In a few experiments where complete depletion was confirmed by FACS, a corresponding complete loss of protection was observed. We have recently shown that 0.5-1.0rag of purified anti-CD8 mAb administered six times is needed to completely deplete CD8+ T cells in each mouse (unpublished results); in the studies here, we have infused lower amounts of mAb. Nevetheless, when in vitro and in t,ivo depletion studies are considered together, it may be concluded that CD8+ T cells primarily mediate clearance of virus while CD4 + T cells may play a minor role in virus clearance4L46. Recent studies have suggested that mice deficient in MHC class I CD8 + CTLs could clear influenza virus infection45 48, while another study showed that similar mice exhibited delayed viral clearance and increased mortality after influenza virus infection49. Even though adoptively transferred T-cell clones have been shown to clear influenza virus infection effectively42'48, uncloned virus-specific CD4 + T cells do not seem to exhibit similar functions. Evidence has accumulated, however, showing that CD8+ CTL, whether cloned or not, are the major effector cells mediating effective clearance of influenza virus infection in oivo 2 5.42.45.,$6,49. It should be noted that numerous recent reports 5°-59 have demonstrated the capacity of exogenous proteins to stimulate MHC class I-restricted CTL responses and

Influenza A cross-protection with a c h i m e r i c protein: I.N. M b a w u i k e et al.

protection against virus challenge. This is contrary to the previously held view that exogenous antigens that are processed through endosomes instead of through the endoplasmic reticulum or cytoplasm, do not stimulate MHC class I CTL, but instead stimulate only MHC class II-mediated cytotoxicityv'6°. It was previously shown that the CTL determinant recognized on D protein was on the HA 2, but on the NSI portion of the chimeric protein 24. Even though NS 1 is antigenically conserved among influenza A subtypes ~v, and can induce subtype cross-reactive CTI clones 61, its function in this system is to enhance the expression of recombinant HA 2 in E. coli. NS~ may also help to stabilize the tertiary structure of the construct to allow effective presentation of the immunodominant epitope to T cells. In a recent study, a chimeric protein (FG) consisting of the F and G surface glycoproteins which represent the major T and antibody epitopes of human respiratory syncytial virus (RSV), respectively, was constructed in the baculovirus expression system 62. FG was shown to protect cotton rats against challenge with human RSV 63. Using a similar FG chimeric protein, others have determined that the immunodominant T-cell epitope on the F protein of RSV is recognized by human lymphocytes64. It is therefore feasible to recombine T- and B-cell epitopes or other desirable determinants into a chimeric protein for effective immunization. In summary, recombinant influenza A/PR/8/34 (H 1N 1)derived chimeric D protein (NSI 1-81-HA2 65-222) induces protective influenza A-specific MHC class I CTL immunity in outbred as well as inbred mice 26. Cytotoxic CD8 + T cells mediate clearance of virus infection in the absence of neutralizing antibody, although CD4 + T cells are also required. Protection was partially crossreactive among influenza A/H1N1 and A/H2N2 subtypes, but not the A/H3N2 subtype. The duration of protection was long and similar to that induced by live virus infection. These results suggest that D protein, through a conserved sequence on the H A 2 polypeptide, has the potential to induce partially cross-reactive CTL that could protect against influenza A virus disease in an outbred human population.

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ACKNOWLEDGEMENTS The authors are grateful to Annette Galvan and Khiem Pham-Nguyen for technical assistance and to Karen Kincade, Brenda DeVaul and Belinda Felder for typing the manuscript. This study was supported in part by Contract NOI-AI-15103 from the National Institute of Allergy and Infectious Diseases, NIH.

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