CA1073355A

CA1073355A - Influenza vaccine containing purified neuraminidase antigen and method of using the same

Info

Publication number: CA1073355A
Application number: CA243,608A
Authority: CA
Inventors: Edwin D. Kilbourne
Original assignee: Mount Sinai School of Medicine
Current assignee: Icahn School of Medicine at Mount Sinai
Priority date: 1975-01-16
Filing date: 1976-01-15
Publication date: 1980-03-11
Also published as: NL7600443A; AU507149B2; PH13837A; FR2297635A1; BE837603A; AU1016376A; GB1536475A; FR2297635B1

Abstract

SPECIFICATION
ABSTRACT OF THE DISCLOSURE

An influenza vaccine is disclosed which comprises, as an active ingredient, a neuraminidase antigen which has been isolated from an antigenically functional virus and has substantial cross-reactivity with the influenza virus against which the vaccine is to be effective. The neuraminidase antigen can be derived from a virus antigenically representative of the con-temporary or prevalent influenza virus subtype of interest, e.g., H3N2, the Hong Kong variant of influenza.

Description

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The present invention is directed to an influenza vaccine containing an antigenically functional and purified neuraminidase, freed of hemagglutinin protein, which forms the sole active component thereof, and a method of immunization using a vaccine.
The prior art has relied exclusively upon vaccines which comprise, as an active component, viruses containing hemagglutinin and neuraminidase antigens which both have a substantial cross-: . :., :: :. ,: . . : : . .

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~V~3355 reactivity with the type of virus against which the vaccineis to be effective. The term "cross-reactivity" as used herein refers to the ability of a given virus to produce antibodies which will inhibit challenge by another virus. When prior art vaccines are administered to an animal they produce antibodies in the animal which are effective against both surface antigens of the wild influenza virus. The use of such a vaccine protects the host animal against both infection and manifestation of symptoms of illness. However, the protection afforded when such prior art vaccines are used is quite transient for reasons that are not entirely clear to persons of skill in the art.
Various recombinant viruses are known and have been used as laboratory reagents to analyze human and animal sera for their antibody content. In addition, recombinant viruses which contain hemagglutinin and neuraminidase antigens which both have substantial cross-reactivity with the influenza virus which sub-sequently challenges immunity have been used to immunize animals, such as man, swine, horses and fowl. Kilbourne, E.D. "Future Influenza Vaccines and the Use of Genetic Recombinants", Kilbourne, E.D., Bulletin of the World Health Organization, Vol.
41, pp. 643-645 (1969), "Correlated Studies of a Recombinant Influenza Virus Vaccine", The Journal of Infectious Diseases, Vol. 124, No. 5 (1971), and The New York Times, Au~ust 13, 1972.
It is now well established that hemagglutinin and neur- "' aminidase are antigenitically distinct proteins of the envelope of influenza virus, and that by genetic recombination hybrid (recombinant) viruses can be produced in which hemagglutinin is derived from one parental virus, and neuraminidase from the '' ' ' ' ; :..";..;:

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~33S5 other. Such antigenically hybrid viruses have proved useful in the isolation of neuraminidase free of demonstrable hemagglu-tinin protein and in the production of specific antibody to viral neuraminidase.
Although antigenically hybrid recombinants that e~fectively segregate neuraminidase from hemagglutinin antigens have been described, Laver and Kilbourne, Virology, Vol. 30, p. 500 (1966), it has not been recognized heretofore that long-term protection against influenza virus can be obtained by using, in a suitable influenza vaccine, isolated neuraminidase antigen having sub-stantial cross-reactivity with that particular influenza virus of interest as the active ingredient. The use of such a mono-rather than bi- specific antigen in the vaccine allows the animal to become infected with the wild influenza virus with which it is challenged while protecting it against manifesta-tions or symptoms of illness. The presence of such infection in the animal is believed to stimulate the natural immunological response of that animal thereby aiding in giving long-term protection. The use of purified neuraminidase in studies of mice in order to compare the relative effectiveness of specific immunity to viral neuraminidase and immunity to viral hemag~lu-tinin in protecting mice against challenge with influenza virus infection was reported on by Schulman, Khakpour and Kilbourne, Virology, Vol. 2, pp. 778-786 (1968).
The present invention is an influenza vaccine which com-prises, as an active ingredient thereof, purified neuraminidase ,.: , ' : ~, .' , `: , ' ' :'. : .
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antigen having substantial cross-reactivity with the challenge influenza virus against which the vaccine is to be effective.
~hen such a vaccine is administered only antibodies to the neuraminidase of the challenge virus are produced in any substantial amounts. Immunization of the host animal, i.e., those subject to naturally-occurring influenza, is perfo!rmed by administering an effective amount of the vaccine containing the purified neuraminidase antigen of the desired influenza virus by any suitable route of administration, e.g., by intraperitoneal, subcutaneous or intramuscular injection.
Also forming a part of the present invention is an antiyen-ically functional virus derived from parental viruses which are non-neurovirulent and which contain a neuraminidase antigen which has substantial cross-reactivity with contemporary challenge influenza viruses.
The present invention, then, in one aspect, resides in a process for producing a monospecific influenza vaccine for ~ -use in man or other animals subject to infection by natural means by naturally occurring influenza, comprising isolating a neuraminidase antigen from a non-neurovirulent parental virus source as essentially the sole antigenically functional component of said vaccine, said antigen having substantial cross-reactivity with a challenge natural contemporaneous or naturally infective wide type influenza virus, and forming a suitable stable and contaminant-free vaccine therefrom, the concentration of said antigen in said vaccine being sufficient to immunize man or other animals.

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q3355 In another aspect, this invention resides in an injectible monospecific influenza vaccine for use in man or other animals subject to infection by natural means by naturally occurring influenza, comprising a neuraminidase antigen derived from a non-neurovirulent parental virus source as essentially the sole antigenically functional component of said vaccine, said antigen having substantial cross reactivity with a challenge natural contemporaneous or naturally infective wild type influenza virus the concentration of said an~igen in said vaccine being suffi-cient to immunize man or other animals.
The procedure for preparing the antigenically functionalvaccine in which isolated neuraminidase antigen is the sole active ingredient is known in the art and described by Laver, Virology, Vol. 20, pp. 251-262 (1963) and Laver and Kilbourne, Virology, Vol. 30, pp. 493-501 (1966). Basically the process involves disrupting samples of purified recombinant or parental viruses by treatment with sodium dodecyl sulfate (SDS), sepa-rating their protein components by electrophoresis on cellulose acetate and thereafter isolating the specific neuraminidase antigen which has substantial cross-reactivity with the type of virus against which the vaccine is to be effective.
A variety of possible viruses can be used as the source of the purified neuraminidase antigen depending upon the parti-cular strain or strains of influenza virus against which the vaccine is to be effective. It is possible to use quite a few subtype viruses of Type A, or B influenza viruses. The HONl and HlNl subtypes, for example, are described in Bull, ~.H.O.:

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45, 119 (1971). For example, one of the viral parents used in the recombination step can be an influenza virus selected from influenza A subtypes including HONl, H2N2 or H3N2 viruses. One particular virus of great importance at the presentti~e in immunization wor~ is the Hong Kong (HK) variant of influenza which is sometimes designated as the A/Hong Kong ~H3N2) influ-enza virus.
A neuraminidase vaccine according to the present invention which is effective against the Hong Kong variant can be formu-lated by recombining an Ao influenza virus, e.g., A/PR8/34(HONl), with the ~ong Kong variant, e.g., HK/Aichi/68 (~3N2), to form hybrid recombinants, some of which contain hemagglutinin and neuraminidase antigens which both have cross reactivity with the type of influenza virus against which the vaccine is to be effective. ThereaEter the neuraminidase antigen portion of the progeny, eg H3N2, is isolated according to procedure discussed above, said antigen having substantial cross-reactivity with the influenza virus against which the vaccine is to be effective.
Such an antigen ~eg. N2) after isolation, when used as an active component in a vaccine, will protect the host animal against the Hong Kon~ variant of influenza. If the vaccine is desired for use in man, the parent strains used in the initial recombination steps should be non-neurovirulent. The absence of neurovirul-ence in such viruses may be indicated by the inability of the viruses to produce plaques or virus colonies in a human con-junctival cell-culture system (clone 1-5C-4 cells; Suguira et al., Virolo , Vol. 26, pp. 478-488 ~19~5~).

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Table 1 contains data which establishes the non-neurovirulence of the parental virus.
Cqmparative ~itration of X-32 (non-virulent) and X-~ 5 (HK) (neurovirulent)* recombinant viruses in a human ~onjunctival cell line (clone 1-5C-4) (both viruses are anti~enically identical, but X-l5(HR) is dexived from a neurovirulent clrandparent).
dilution plaque virusinoculated formation**
, X-32 <10-0_2 0 lû-4 0 10-g 0 ~ .
X-15~H~)10-~ ~ 10-0 4 ~ .
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Conclusion: two antigenic hybrids, indistinguishable antigenically, but one potentially neurovirulent (i.e., with neurovirulence genes) can be distinguished on the basis of diffexing virulence in a human cell culture system.
The genealogy and derivation of these recombinant antigenically hybrid viruses is presen~ed below:
Derivation of X-15(HK) (Summar~) . . -Ao~NWS/(MX) X HK/16/68 A/NWS/33(HO~l) A/HongKong/1/68(H3N2) (neurovirulent) ..
Ao (E) HK X A/equil .
.. .. (HON2) ¦ HeqlNeql .
X l5(HK) ~leqlN2 erivation o X-32 (Summary) Ao/PR8j34*** X HK/Alchi/68***
A/PR8/AnnArbor/34(HONl) ¦ A/Aichi/68(H3N2) X-31*** X A/Eq/1 ~3N2 ¦ HeqlNeql ~eqlN2 ~ potentially neurovirulent ** focal destruction and lesions of cell culture monolayers ***strain used in commercial vaccine produetion in USA

~0~^~3355 The process used in the ~ormation of recombinant viruses is a known procedure in the art and is described in a number of publications including the following: Kilbourne and Murphy, J. Exper. Med.: 111, 387 (1960); Kilbourne, Science, Vol. 160, April 5, 1968, pp. 74-75; and ~aver and Kilbourne, Virology, Vol. 30, pp. 493-501 ~1966). It basically comprises inoculating chick embryo allantoic sacs with ~he two viruses which are to be recombined. The resultant hybrid progeny are hybrids of both parental viruses. Generally, a high yield, i.e., high growth poten~ial, and a low yield virus will be used as parents. A portion o~ the hybridized progeny of the recombination step can be eliminated in a cloning step by adding an antibody which is specifically cross-reactive with the high-yiela parental virus and h~brid pro~eny possessing the hemagglutinin o~ that parent. The remaining hybrid progeny are then, if necessax~
inoculated into further chic~ embryo allantoic sacs, and when removed, contain a higher percentage o~ ~irus o~ the desired -antigenic composition. Diluting the viruses to high dilution values insures that the high yield virus progeny will outgrow - .:
those having the lower growth potential.

The dosage of the neuraminidase antigen thàt is ... . . . . . .... . . .
administered to man is equivalent to that contained by whole viruses comprising 500 to 1000 chick cell agglutinating units The present invention is further illustrated by the ~ollowing examples:
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Example I ~-. . . . . , -To produce the neuraminidase antigen usea in the present ., .' . ' ~ '.. ' ''' ' ' ' ' ~ ':
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invention neuraminidase is isolated in active form ~ia an affinity column ~earing an inhibitor speciia for this component.
The column, developed ~y Cuatrecasas and Illiano, Bi _ m Res. Cummun., 44: 178 (1971), is prepared by CNBr activation ~f agarose, addition of the tripeptide, gly-gly-tyr, and diazotization of the inhibitor, N-p-aminophenyl oxamic acid. This column isolation technique was adapted for use with influenza virus by the addition of "Trlton x-lOO~* to absorbing and eluting buffers, Bucher, Abstr. Amer~ Soc. Microbiol, p. 215 (1973), The use of "Triton X-l~0" both stabilizes and maintains the solubility of ~he neuraminida~e. The dissociation step of the i~tact virions is Lmpcrtant, i~ 5DS alone is used as the dissociating a~ent, the neuraminidase is ~uickly inactivated on adjustment to a mildly acidic pH 5.0 even for the relatively hardy X-7 viru~ strain. I
"Triton X-100" is used as the disrupting agent alone, the NA does not dissociate from HA, although the NA retains its activity. The use of the two detergents in sequence ~1% SDS at pH 7.0, then "Triton X-100" to 10~ concentration followed by adjustment to pH 5.O) insures that the virio~ will be fully dissociated but activity will remain. After dialysis of ~he viral protein preparation at pH 5.0, the neuraminidase is absorbed to the column with the absorbing, pH 5. 0, buffer. All other proteins pass through the column unhindered. The neuraminidase is eluted with pH 9.1 buffer, generally in a peak with a center of about pH 7, All fractions are assayed for protein, Lowry et al., J. Biol. Chem. 193: ~65 (1951), and for neuraminidase activity, ... .. .. ..
Cassidy et al., Methods in Enzymology, Vol. 8, pp. 680-685 (1966~. The neuraminidase purification had re3ultant yields of 125~ of the activity of the disrupted .

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1~33~5 ~iral preparation associated with 1.6% of total protein. The protein recovery was 60.6~. The increase in specific activity was 77 fold. Results of the chromatogram are summarized in Table 2~ On polyacrylamide gel electrophoresis the neuraminidase migrated as a single high molecular weight component (molecular weight of about 250,000~ when 10 ~g material was applied to the gel~ af er a single cycle bf purification ~
This procedure was also applied to a B strain (from an outdated lot of vaccine) and to a prepaxati~n of X 31, These strains, X-31 and B/Mass, have contami-nating proteins accompanying them on the gels,. at l~wer levels, but modLfications of the aisrupting techni~ue shoula re~ult in greater purity or these strains. It has been found that a second cycle on the af~inity column after the addition of a relatively low level of reducer (0.01 M dithiothreitol) results in elimination of conta~inating polypeptides for ~he X-31 and B
strai~s. The methodology was elaborated primarily for the X-7 strain and found to work with other strains. Excellent recovery of active neuraminidase can be achieved at this step ~or all 0 strains attempted including the very unstable A~ neuraminidases~
Example II
A ~accine was produced containi~g the purified neuraminidase antigen produced in Example I by inoculating chick embryo allantoic sacs with the diluted virus and harves ing the allantoic fluid after a two-day growth period. The allantoic fluid was preliminarily purified by low speed centrifugation which was followed thereafter by zonal ultracentrifugation to produce a semi-purified allantoic fluid ~iru5. A pr~duct was produced consisting chiefly of puriied influenza virus which was then subjected to disruption with detergents and a~finity chromato-graphy for the isolation of purified neuraminidase as described in Example I.

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Immunization of mice with isolated X-7(Fl)lN21 ~euraminidase. Mice were injected subcutaneously with 0.3 ml o a mixture o e~ual parts of ~aline and complete Freund's S adjuvant or of a 1:16 dilution of isolated N;2 enzyme (E) de-rived from X-7(Fl) (HON2) and adjuvant. The injections were ~epeated 40 days later, and 7 day~ after the booster injection three mic2 from each group were ~led. Serum antibody levels were ~easuxed in ~I tests agai~3~ Jap~ 305 (~2N2), X~7(~1) r (~ON2), X-3(~2Nl), and ~-lS (HeqlN2) viruses. Enzyme-inhibitio~
titexs were determined with X-7(Fl) (HON2) virus, and pla~ue size-reduci~g titers were determined against X-7 (HON2~ ~nd NWS ~ONl) vixuses in clone 1-5C-4 human conjuncti~al cells ~Table 3). Immunization of mice with E neuraminidase did not induce production of HI antibody against any of the viruse~
tested except X-15, but it did produce serum titers o 1:400 o~ enzyme-inhibiting antibody against X-7(Fl)[N2~ enzyme.
In addition, the pooled sera of mice immunized with puri~ied (E~ enzyme had plague size-reducing activity against X-7 virus (~ON2) but not against NWS (HONl) -- a virus that differs ~rom the X-7 viru~ recombinant only in havin~ a diferent neuramini- -dase pxotein (Nl).
When groups of mice similarly immunized were challenged with Jap. 30~ (H2N2) virus, the results given in Table 4 were obtained. Mice immunized with (E) neuraminidase had consider-ably lower pulmonary vixus titers thhn control mice 2 and 4 days after Jap. 305 virus challenge; they had less extensive lung lesions 7 days after challenge.

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Effect of immunization with isolated ~2 neuraminidase on influenza Jap. 305 ~H2~2) virus infection of mice , _. . _ _ . _~
. Pulmonary Lung Immuniza~iona ~irus titersb le ions (%) Day 2 Day 4 __ _ . , _ _ ,.
Saline-adjuvant 0............. 7.8 7.2 44.0 Enzyme-adjuvanta... ~O......... 5.5 <3.7 5~0 ~ ._ _ .
aSubcutaneous injection as in Table 3.
bEIDa R ~ log10, five animal~ in each group.
Enzyme - neuraminidase .

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In addition~ enzyme (N2) immunized and unimmunized mice were ch~llenged with X-7(Fl) tHON2), X-15 (HeglN2), NWS
(HONl), and X-9 (~2Nl~ viruses. Titers of pulmo~ary virus 3 days after chailenge are given in Table 5. Significant re-ductions of pulmonary virus titers were observed when miceimmunized with ~N2) neuraminidase were challenged with viruse~
that-contain (N2) enzyme 1X-7(Fl), X-151 but not whe~ they ~ere challenged with viruses that have an antigenically dif-ferent neurami~idase ~Nl) protein, e.g., (NWS, X-9).
Thus, immunization of mice with isolated N2 neuraminidase resulted in enzyme-inhibiting serum antibody to N2 neuraminidase and, despite the lack of serum an~ibody reactive in hemagglutinatio~-inhibition tests with any o the challenge ~iruses except X-15, led to significant protection against challenge infection wi~h viruses containing N2 neuraminidase.
The code-named ~iruses used i~ forming the purifled neuraminidase antigen and vaccine of the present invention can be obtained from ~he Bureau of Biological Standards o~ the Food and Drug Administration.

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-10'~3355 T~BLE 5 .

Effect of immunizations with purified N2 neuraminidase on challenge infections with in~luenza viruses which do and do not contain N2 enzyme ~E) Pu~onary virus titers 72 hours I~munizat~ona ater challengeb ____ j__ . X~75Pl) NWS X-9 X~15 (~0~2) ~ONl) (H2Nl) ~HeqlN2~-~ j _ _ ._ ,_~
Saline-adjuvant .................... 5.1 6.8 6~0 607 Enzyme-adjuvant .. ~............... <2~1 7.0 6.4 4.4 _ . _ _ . . _ ~ ...... --aSubcutaneous injection as in Table 3.
bEIDso~ log10~ five animals in each group.

A person of skill in the art upon reading the .
foregoing will become aware of a number of modiications which can be ~ade to the invention described above without departing from the spirit and scope thereof. ~ence, the foregoing is not.
to be taken as limiting since it is intended to be me~ely illustrative o a number of embodiments of the invention.
The appended claims define the scope o~ protection sought.

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for producing a monospecific influenza vaccine for use in man or other animals subject to infection by natural means by naturally occurring influenza, comprising isolating a neuraminidase antigen from a non-neurovirulent parental virus source as essentially the sole antigenically functional component of said vaccine, said antigen having substantial cross reactivity with a challenge natural con-temporaneous or naturally infective wild type influenza virus, and forming a suitable stable and contaminant-free vaccine therefrom, the concentration of said antigen in said vaccine being sufficient to immunize man or other animals.

2. A process according to claim 1, wherein the neuraminidase antigen is derived from an influenza virus selected from the group consisting of a subtype of Type A or B influenza viruses.

3. A process according to claim 1, wherein the neuraminidase antigen is derived from the Hong Kong (H3N2) variant of Type A influenza virus.

4. A process according to claim 1 wherein the vaccine is formed so as to provide a concentration of said neura-minidase antigen in said vaccine sufficient to provide a unit dosage equivalent to that contained by whole virus comprising about 500 to 1000 chick cell agglutinating units.

5. An injectible monospecific influenza vaccine for use in man or other animals subject to infection by natural means by naturally occurring influenza, comprising a neuraminidase antigen derived from a non-neurovirulent parental virus source as essentially the sole antigenically functional component of said vaccine, said antigen having substantially cross reactivity with a challenge natural contemporaneous or naturally infective wild type influenza virus, the concentration of said antigen in said vaccine being sufficient to immunize man or other animals, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

6. An injectible monospecific influenza vaccine for use in man or other animals, wherein the neuraminidase antigen is derived from an influenza selected from the group consisting of a subtype of Type A or B influenza viruses, when prepared by the process of claim 2 or by an obvious chemical equivalent thereof.

7. An injectible monospecific influenza vaccine for use in man or other animals wherein said neuraminidase antigen is derived from the Hong Kong (H3N2) variant of Type A
influenza virus, when prepared by the process of claim 3 or by an obvious chemical equivalent thereof.

8. An injectible monospecific influenza vaccine for use in man or other animals wherein the concentration of said neuraminidase antigen in said vaccine is sufficient to provide a unit dosage equivalent to that contained by whole virus comprising about 500 to 1000 chick cell agglutinating units, when prepared by the process of claim 4 or by an obvious chemical equivalent thereof.