Murine model for evaluation of protective immunity to influenza virus

Miroslav Novak*$, Zina Moldoveanu and Richard W. Clompanst

*+, Dennis P. Schafer*, Jiri Mesteckyt

We have characterized a murine model as an inexpensive, readily available and sensitive animal model for the evaluation of protective immune responses induced by various routes of administration of injluenza A vaccine preparations. Using a non-mouse-adapted human influenza virus to ircfect unanaesthetized animals intranasally, we established that the optimum dose for in&ction of Balblc mice was lo4 plaque forming units of virus and that the optimum sampling time for measurement of virus yields in the organs of the respiratory tract was 72 h after challenge. We found that the infection was initiated in the nose and progressed by descending into the trachea and lungs over a period of days. Evaluation of protection against iqfection clearly showed that the tissues of the mouse respiratory tract were completely protected after administration of whole killed virus intranasally andpartly protected when virus was administered subcutaneously. The protection correlated with the level cjf virus-speciJic IgA antibodies in saliva.

Keywords: Mouse model; influenza A virus; mucosal immunity; protection

INTRODUCTION

Efforts to provide improved methods for immunization against influenza are currently impeded by the lack of a widely available, inexpensive animal model for the direct evaluation of protection against challenge infection. Ferrets are a well established model of influenza infection’-4 and squirrel monkeys have been used successfully to study influenza and other respiratory tract infections5-‘. They can be infected with human strains of influenza virus and display a mild disease that is similar to human infections. However, the high cost of purchase and care of these animals make the large-scale evaluation of alternative vaccines an expensive endeavour; further- more, the reagents required for assessment of the mucosal immune response are not readily available. In contrast, mice require the lowest costs for animal purchase and care. Animals and reagents are readily available, and mice are a well established model for studying both systemic and mucosal immune responses8*9. However, human influenza viruses do not cause an overt disease in mice. Serial passage of human viruses through the mouse lung results in a lethal infection in the lower respiratory tract within days of infection with such

*Secretech Inc., 1025 18th Street South, Suite 201, Birmingham, Alabama 35205, USA. +Department of Micro- biology, The University of #Alabama at Birmingham, Birmingham, Alabama 35294, USA. ~To whom corres- pondendence should be addressed. (Received 3 January 1992; revised 11 May 1992; accepted 11 May 1992)

mouse-adapted strains”-’ 3. The inoculum is usually instilled directly into the lung, which is in the immunological domain of IgG derived from the circulation14. This model is therefore acceptable for the evaluation of systemically administered influenza vaccines. However, it cannot be used reliably to evaluate the role of mucosal immunity in protection against infection, as the inoculum bypasses the mucosal surfaces of the upper respiratory tract.

Iida and Bang” suggested that an experimental infection in mice could be restricted to the nasal passage without concurrent infection in the lower respiratory tract. This observation was extended by Yetter and co-workers3, who used a radioiodinated protein solution to compare the distribution of an inoculum in anaesthetized compared with unanaesthetized mice. The inoculum, administered in a small volume intranasally to unanaesthetized mice, was restricted to the nasal epithelium. Ramphal and co-workers16 successfully infected mice intranasally with non-mouse-adapted influenza virus, and although the infection produced no obvious disease symptoms, virus replication could be assayed by virus titration.

In the present study, we have examined the disease process in mice infected intranasally with non-adapted influenza virus with respect to the dose response, the time course of the response, the sequence of organ involvement, and the suitability of the model for the evaluation of mucosal and systemic protective immune responses induced by intranasal and subcutaneous immunization, respectively.

0264410x/93/01005 0 1993 Butterworth-Heinernann Ltd Vaccine, Vol. 11, Issue 1, 1993 55

Mane model for mfluenza virus mfechon. M Novak tl~ jj

MATERIALS AND METHODS

Mice Plaque assay fur virus titration

LI.Cw-MKL irhesus monkev kldnev t cells wx

t-emale Balb/c mice, 6 -8 weeks old. purchased from Charles River Breeding Laboratories Inc. (Wilmington, MA. USA) were used in all experiments. These mice were housed five or six to a cage and the care and use of these animals followed National Institutes of Health Guidelines.

Virus

The influenza virus A/Udorn/307/72 (H3N2), BK6, Egg 3, clone 3A (7-25-89) was a gift from Dr B.R. Murphy (National Institutes of Health, Bethesda, MD, IJSA). This virus was passaged once in embryonated chicken eggs and the allantoic fluid stored as stock virus (infection titre 2.53 x 10’ plaque forming units (p.f.u. ) per 0.2 ml) at - 130°C. Working dilutions for infection of mice contained 103- lo6 p.f.u. per 10 ~1, prepared in sterile Dulbecco’s medium supplemented with 1% bovine serum albumin and frozen in aliquots of 0.2 ml in sterile Eppendorf tubes at - 130°C.

Virus preparation

Ten-day-old embryonated chicken eggs (400- 500 ) were infected with 0.1 ml of stock virus diluted 1: 1000 in L-l 5 medium supplemented with SPG (2.18 M sucrose, 0.038 M KH,PO,, 0.072 M K,HPO, and 0.049 M monosodium glutamate). After incubation for 48 h at 35-36”C, the allantoic fluid was collected and clarified by centrifugation at 37OOg,, for 20 min. The virus was then collected from the supernatant by centrifugation at lOOOOOg,, for 45 min. The pellets were left overnight in 0.5 ml phosphate-buffered saline (PBS) at o”C, diluted to 6 ml with PBS, vigorously mixed and aggregates removed by centrifugation at 13OOg,, for 15 min. This procedure was repeated three times. After the third centrifugation, the pellet was sonicated and again centrifuged. The pooled supernatants containing the virus suspension were loaded on top of lo-60% continuous sucrose gradients in PBS and centrifuged for 2 h at lOOOOOg,, in a swing-out rotor. The virus bands were collected, diluted 1: 1 in PBS and the virus inactivated by incubation with formalin (1:4000 v/v) for 72 h at 37°C. The material was dialysed overnight at 4’ C against PBS, pelleted as above, resuspended in PBS or water to 5 mg protein ml - ’ and stored at - 80°C. Protein was measured by a Coomassie blue binding assay (Pierce, Rockford, IL, USA) of sodium hydroxide-disrupted virus.

Challenge infection of mice

For determination of antigen-specific antibodies ELBA was performed in 96-well polystyrene microtitre plates (Dynatech, Alexandria, VA, USA) coated with purified A/Udorn influenza virus at a concentration of 4 pg ml- ‘. Endpoint titres of serum and saliva were determined using horseradish peroxidase-labelled goat IgG against mouse Ig or IgA (Southern Biotechnology Associates, Birmingham, AL, USA) and substrate 2,2’- azino-bis-( 3-ethylbenzthiazoline) sulphonic acid (Sigma, St Louis, MO, USA). The colour developed was measured in a Vmax photometer (Molecular Devices, Palo Alto, CA, USA) at 414 nm.

Unanaesthetized mice were infected intranasally. Haemagglutination inhibition (HI ) reaction was During the procedure the upper part of the nose was performed with mouse sera diluted 1:5 with PBS and held down to minimize the possibility that the virus would treated for removing non-specific inhibitors (heated at be swallowed or enter the trachea directly. The mice were 56°C for 30 min; incubated with 25% acid-treated kaolin then killed at the stated periods. The noses, tracheas and for 30min; and incubated with 10% suspension of lungs were collected separately and homogenized in L- 15 chicken red blood cells for 30 min). Twofold dilutions of medium supplemented with SPG, antibiotics (300 U sera were prepared in 96-well microtitre plates. Viral pencillin and 300 pg streptomycin ml-’ ) and 50 pg suspension (8 HA units in an equal volume) was added Fungisone ml- ‘. Noses and tracheas were homogenized to each well and incubated at room temperature for with washed, sterile sand in a mortar with pestle. Lungs 30 min. A 0.5% suspension of chicken erythrocytes was were homogenized in a Potter-Elvehjem tissue grinder. added to each well and incubated at room temperature Homogenates were frozen separately in sterile tubes at for 45--60 min. The HI titres were expresssed as the -80°C for later titration of virus recovery using the reciprocal of the highest dilution that completely plaque assay. inhibited haemagglutination of erythrocytes.

obtained from the American ‘Type Culture Collectlc-tn i Rockviile, MD. USA i and maintained m Dulbecco % medium supplemented with 10% bovine calf serum and antibiotics, Confluent monolayers in six-well plates were washed twice with PBS, inoculated with 0.3 ml of the appropriately diluted virus and incubated for 1 h at 37 C‘ with tilting every 15 min. Unabsorbed virus was removed by washing the plates with 0.5 ml PBS and the cells overlaid with 0.9S; agar contaming trypsin (2 pg trypsm ml-‘) in Dulbecco’s medium supplemented with antibiotics. Plaques were counted at day 3 and day 4, Results are expressed as the arithmetic mean of influenza virus from the whole organs of five to ten animals.

Immunization of mice

Mice were immunized intranasally using the above procedure for challenging of mice with formalin-fixed A/Udorn virus (50 pg in 10 ~1 PBS) and subcutaneously using 0.1. 1.0, 10, and 50 pg in 100~1 PBS.

Collection of biological samples

Blood was collected from the tail veins of mice before and at selected times after immunization. Blood was centrifuged and plasma was collected and frozen. Stimulated saliva was collected with capillary tubes after intraperitoneal injection of mice with carbamyl-choline chloride (2 pg/mouse). Amounts of 2 pg each of soybean trypsin inhibitor, phenylmethylsulphonyl fluoride, sodium azide and fetal calf serum were added before clarification and storage at -80°C.

Antibody assays

56 Vaccine, Vol. 11, Issue 1, 1993

Murine model for influenza virus infection: M. Novak et al.

RESULTS

Time course of virus infection

Experiments were designed to determine the time course and tissue dilstribution of virus infection. Initially, we infected mice with 1 x lo6 p.f.u. of A/Udorn virus (Figure 1). At 10 min or 4 h postinfection, only low levels of virus could be rec:overed, and the virus was found only in the nose. Thus, the higher titres of virus recovered at later times from the nose and other tissues were a result of virus replication and not due to residual inoculum of the virus.

Virus titres in the nose reached peak levels at day 1 and subsequently declined, but remained at detectable levels as late as 5 days postinfection. In contrast, virus titres in the trachea were barely detectable on day 1, reached a peak on days 3 and 4, and declined markedly by day 5. In the lu:ng, low levels of virus were detected on day 2, and peak: titres were not obtained until days 4 and 5 postinfection.

In a separate experiment, in which virus titres were measured for as long as 8 days postinfection, nasal titres continued to decline but remained at significant levels; virus was cleared from both tracheas and lungs by day 7 in all animals.

These results suggest that a progressive infection occurs, which is initiated in the nasal tissues, then progresses to the trachea and finally to the lungs.

Dose response

To determine the optimal dose for intranasal infection, groups of six mice were inoculated with 103-lo6 p.f.u. of virus. At 3 da.ys postinfection, virus titres were measured in homogenates of noses, tracheas and lungs

3 4 5

Time after infection (days)

Figure 1 Virus recovery from mice following intranasal infection with 108 p.f.u. of influenza A/Udorn (H3N2) virus. Mice were infected intranasally with 106 p.f.u. of influenza virus in 10 ~1 of Dulbecco’s medium supplemented with 1% bovine calf serum (BCS). Mice were killed after 10 min (five mice), 4 h (five mice); 24 h (five mice); 48 h (five mice); 72 h (six mice); 96 h (six mice); or 120 h (five mice), and from each mouse the nasal tissue (nose, maxilla, cranium), trachea and lung were harvested. Noses and lungs were homogenized individually at 10% w/w in L-15 medium supplemented with 1% 10 x SPG (Sucrose, 21.8 M; KH,PO,, 0.039 M; K,HW,, 0.072 M; monosodium glutamate, 0.049 M). Tracheas were homogenized individually in 1 ml of medium (as above). Virus recovery was measured as p.f.u. on LLC cells in six-well plates. Each bar on the graph represents the arithmetic mean of influenza virus from whole organs of five or six mice. n , Nose; 8. trachea; 0, lung

10'

Infection dose per mouse (p.f.u.1

Figure 2 Virus recovery from mice following intranasal infection with different doses of influenza A/Udorn. Groups of six mice were infected intranasally with influenza virus (allantoic fluid, diluted appropriately with Dulbecco’s medium supplemented with 1% BCS) in doses of loJ, l(r. 1oJ and 106 p.f.u. per mouse in 10~1. After 72 h mice were killed and noses, tracheas and lungs were homogenized and titrated as described in the legend for Figure 7. n , Nose; 0, trachea; 0, lung

(Figure 2). Each virus dose tested was found to result in detectable infection. However, the tissue distribution of virus at 3 days varied with the input dose. At an input dose of lo3 p.f.u., virus was recovered predominantly from the nose, with low levels occurring in either the tracheas or the lungs. In contrast, at higher inoculum doses, the nasal titres were somewhat reduced, but increased amounts of virus were recovered in tracheas or lungs. It is not known why the virus yields were reduced at higher inoculum doses, but this could possibly be the result of defective interfering particles.

Time course of infection at lower dose

The lowest dose at which a significant infection occurred in each tissue was lo4 p.f.u. The time course of the infection with an input dose of lo4 p.f.u. was therefore determined (Figure 3 ). As a comparison of Figures I and 3 shows, some differences were evident in the results. Nonetheless, the general pattern of infection was similar, with virus titres peaking initially in the nose (days 1 and 2) and later in the trachea (day 3); virus titres in the lungs were significantly lower and peaked on day 4.

Reproducibility

Based on these results, we decided to use a time of harvest at 72 h, and an inoculum dose of lo4 p.f.u. in subsequent challenge experiments designed to measure vaccine efficacy. It was anticipated that this time point would reveal significant differences in virus titres in the nose and trachea between groups of immunized compared with non-immunized animals, and possibly in the lungs as well.

Titres were compared from six groups of mice which were infected in separate experiments on different dates to test the reproducibility of the model (Figure 4). All the mice were infected with lo4 p.f.u. and were killed 72 h postinfection. While some interexperimental vari- ability could be observed, the general pattern of infection

Vaccine, Vol. 11, Issue 1, 1993 57

blurme model for influenza virus intecbon M. Novak CT i:

- lo5

2 d ci

? lo4 c

Lo : .-

> lo3

lo2 1 ?

Days after infection

Figure 3 Virus recovery from mice followmg intranasal infection with lo4 p.f.u. per mouse of influenza A/Udorn. The experiment differs from that shown in Figure 7 principally in that the infectious dose was lo4 p.f.u. m, Nose; 0, trachea; 0. lung

lo3 1 2 3 4 5 6

Control group

Figure 4 Virus recovery from six different groups of mice following intranasal infection with l(r p.f.u. of influenza A/Udorn. Groups of mice (five to ten in each group) were infected intranasally at age 8-12 weeks with 104 p.f.u. of influenza per mouse in six separate experiments at different times. Mice were killed 72 h postinfection and their organs were assayed for recovery of virus. n , Nose; 8, trachea; 0, lung

remained constant. Particularly, these data indicated that the highest level of virus was consistently found in the trachea. The average level of infectious virus particles recovered per gram of tracheal tissue was 1.2 x lo7 p.f.u., nearly 50 times higher than the yield per gram of nasal tissue and more than 200 times higher than the yield from lung tissue.

Immunization and challenge

To confirm the utility of this disease model in the evaluation of vaccine efficacy, we tested the ability of a high dose of whole, formalin-inactivated influenza vaccine administered either by the conventional paren- teral route (subcutaneously) or by a mucosal route (intranasally ) to protect against challenge infection.

Mice were immunized twice (day 0 and day 28), with a total of 100 pg of virus (50 pug x 2), and were challenged

it11 da\, ifi s f E(/LIW .” /. ~Attcl >ubclrl;tnWus :rrlmunlzatloil.

the i&p< MCI-~’ completely protected and only [race levcl~~ ~~I’viru, were recovered ;II the trachea (of ten mice in thtb group o111\ i)ne had rirus iri II\ trachea,. In now. protectioti L\s;is noi z~~mplelc. Out the diliOliI!t c)f iif 12, recovered \‘~‘a\ reduced nearly tenfold. [ntransal immurii- /ation ulduccd high !cvCi S ji scCrctcri1 ,.mtibodks ! 7‘ah/c i I and 3 days :J’ter the challenge ail three tissuc~ were completely clear l)i‘ virus. This immunization rotite induced !he highest levels of trrus-spcclhc fpA in the saliva.

To evalualc the ability of Ihe modei tcj difFerentiaie between degrees of protection afforded by higher and lower doses of vaccine. we conducted a second experiment. in which mice were immumzed only once subcutaneously with ;i dose of 0.1 ,~g, 1 .O ,~g 01 10.0 LLg of virus protein (Figurc~ 6 1. Virus titres decreased

105

; lo4 ,’ d.

a, h

lo3 .-

G .f: lo2 >

10’

,“O PBS [S.C. i Antigen is.c.1 Antigen (i.n.1

immunogen (route of Immunization]

Figure 5 Virus recovery from control versus immunized mice. Groups of ten mice were injected subcutaneously with PBS or with whole formalin-fixed virus and intranasally with whole formalin-fixed virus (5Opg of virus protein per dose) on day 0 and day 28. Mice were challenged at day 56, killed 72 h later and noses, tracheas and lungs were processed for recovery of virus by plaque assay. n , Nose; a, trachea; m, lung

80

F 70

=i- 2 60 x

; 50 c’ 6. - 40

2

3 30

2 A > 20

10

0 PBS 0.1 1 .o 10

Dose of antigen (fig)

Figure 6 Virus recovery from unimmunized mice and mice immunized subcutaneously with varying doses of influenza virus. Mice were inoculated with a single dose of PBS or 0.1 pg, 1 pg or 10 fig of influenza virus. n , Nose: U, trachea; 0. lung

58 Vaccine, Vol. 11, Issue 1, 1993

Table 1 ELISA and HI antibody response to A/Udorn immunization of mice (correlate with figure 5)

End point titre

Serum Saliva Route of - immunization ELISA lg HI ELISA lg ELISA IgA

Non-immunized 9 850/l .48 20.5/5.3 0 712.3 Subcutaneous 194000/2.3 10/0.4 11/4.1 1213.2 lntranasal 111000/1.3 15 18411.7 640/1.9

*Geometric mean standard deviation

in a dose-dependent manner as the antigen dose decreased. In tracheas, virus was presept at doses of 0.1 pg and 1 .O pg (only one mouse from five in each group) but not at the 10.0 pug dose. The relationship was clearest in the nose, where virus was present at all three vaccine doses, but where viral load declined almost linearly with vaccine dose. The overall pattern in all three tissues is consistent with the hypothesis that the conventional, parenterally administered influenza virus vaccine protects best in the lungs, less well in trachea and only partially in the nose.

DISCUSSION

Several characteristiscs of this model are important to the efficient, valid evaluation of candidate vaccines and immunization routes that induce protective immune responses against influenza infection. The progress of infection is analogous to human influenza infection, with initiation in the nose and descent over a period of days into the trachea and1 lungs.

Protection against infection can be evaluated in this model in a dose-dependent manner. There was a clear dose-response relationship in the nose, and to a lesser extent in the lung and tracheas. If the study were to be extended to even lower subcutaneous doses, the dose- response relationship in lungs and tracheas might be more clearly established. Similar experiments also might establish a dose-response relationship for mucosal vaccines. This poirrt is highly relevant because of the recently re-emphasin:ed importance of local immunity in the protection of mucosal surfaces as the most important portals of entry of microbial pathogens17*‘8.

Unlike other murine infection models that use mouse-adapted viruses, this model allows flexibility in the virus strain used for immunization and challenge. The ability of a variety of human viruses to infect mice has been established previously by other authors’0~‘3’19-21. In this study, we have used a human strain, A/Udorn, because it has been used successfully in a squirrel monkey model by other investigators**. Thus, use of this strain simplifies the sequential testing of vaccine protocols in mice, monkeys and humans.

A disadvantage of the model is the variability of the virus titres in infected tissues. This variability has also been experienced by others (P.A. Small, personal communication). In addition, a lack of reproducibility of the frequency of lung infections was observed. A lung infection was never observed in an animal in the absence of infection in the trachea. However, high titres of virus in the trachea did not always lead to infection in the lung. These problems could be overcome by the use of

Murine model for influenza virus infection: M. Novak et al.

mouse-adapted virus produced by serial passage of virus through the lung, by the use of an increased inoculum dose or by anaesthetizing the mice prior to challenge. However, each of these strategies would reduce the value of the model by decreasing its analogy to the human disease.

We do not view the lack of reproducibility of infections in the lung as a critical problem. In our opinion, the trachea, which is protected by both serum IgG and mucosal IgA antibodies, is of greater relevance in the evaluation of protection against serious consequences of disease. Recent strains of influenza virus have infected tracheas and bronchial passages with greater frequency than lungs. In those tissues, which are a primary target of the influenza virus, the virus destroys the ciliated epithelium, and thus inactivates the mechanical barrier against adventitious pneumonia infection of the lungs by bacteria present in the normal flora of the upper respiratory tract23. Therefore, in this regard, the mouse infection is a particularly close model of the human infection16.

The mouse is generally regarded as a good immunological model for which the reagents necessary for accurate qualitative and quantitative analysis of both the systemic and mucosal immune response are readily available. The ability to establish a progressive human-like infection using influenza virus strains not adapted in the mouse renders it a useful, inexpensive disease model for influenza infection.

ACKNOWLEDGEMENTS

The authors are grateful to Fiona Hunter for editorial assistance and Joellynn Heaton for preparation of the manuscript. The research was supported in part by Grant no. 1 R43 AI31008-01 from the Department of Health and Human Services, Small Business Innovation Research Program awarded to Secretech, Inc.

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13

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