NZ620275B2 - Recombinant swine influenza virus and uses thereof - Google Patents
Recombinant swine influenza virus and uses thereof Download PDFInfo
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- NZ620275B2 NZ620275B2 NZ620275A NZ62027512A NZ620275B2 NZ 620275 B2 NZ620275 B2 NZ 620275B2 NZ 620275 A NZ620275 A NZ 620275A NZ 62027512 A NZ62027512 A NZ 62027512A NZ 620275 B2 NZ620275 B2 NZ 620275B2
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- swine
- influenza
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Abstract
Disclosed is a recombinant, chimeric influenza A virus comprising eight segments, wherein said virus comprises more than one haemagglutinin (“HA”) segment (“segment 4”) from more than one influenza subtype, wherein said virus comprises segments 1-5, 7 and 8 from a porcine H1N1 influenza subtype and a second segment 4 from a porcine H3N2 influenza subtype, wherein the H3N2 segment 4 comprises neuraminidase (“NA”) packaging sequences from said H1N1 influenza subtype located 3’ to said second segment 4, and further wherein all or a portion of the NA segment (“segment 6”) of the H1N1 influenza subtype is missing to render an attenuated virus. Also disclosed is a recombinant construct comprising: (a) a porcine influenza H3N2 subtype HA segment; and (b) porcine influenza H1N1 subtype NA packaging sequences located 3’ and 5’ to said H3N2 HA segment, wherein the construct lacks all or a portion of the NA coding sequence of the H1N1 subtype. a second segment 4 from a porcine H3N2 influenza subtype, wherein the H3N2 segment 4 comprises neuraminidase (“NA”) packaging sequences from said H1N1 influenza subtype located 3’ to said second segment 4, and further wherein all or a portion of the NA segment (“segment 6”) of the H1N1 influenza subtype is missing to render an attenuated virus. Also disclosed is a recombinant construct comprising: (a) a porcine influenza H3N2 subtype HA segment; and (b) porcine influenza H1N1 subtype NA packaging sequences located 3’ and 5’ to said H3N2 HA segment, wherein the construct lacks all or a portion of the NA coding sequence of the H1N1 subtype.
Description
RECOMBINANT SWINE INFLUENZA VIRUS AND USES THEREOF
TECHNICAL FIELD
The present invention pertains generally to influenza virus and immunogenic
compositions and methods for treating and preventing influenza infection. In particular,
the invention relates to recombinant, chimeric swine influenza viruses expressing more
than one hemagglutinin (HA) subtype.
BACKGROUND
Swine influenza (SI) is an acute respiratory disease of swine caused by type A
and type C influenza viruses. Influenza A viruses are segmented negative-strand RNA
viruses and can be isolated from a number of other animal host species, including birds,
humans, horses, whales, and mink. Although whole influenza viruses rarely cross the
species barrier, gene segments can cross this barrier through the process of genetic
reassortment, or genetic shift. Pigs support the replication of both human and avian
influenza A viruses and have been postulated to play an important role in interspecies
transmission by acting as a “mixing vessel” for reassortment between viruses specific to
different host species (Scholtissek, Eur. J. Epidemiol. (1994) 10:455-458). This may
lead to the generation of influenza viruses capable of crossing the species barrier to
humans.
Influenza virions include an internal ribonucleoprotein core (a helical
nucleocapsid) containing the single-stranded RNA genome, and an outer lipoprotein
envelope lined inside by a matrix protein (M1). The genome of influenza A virus
consists of eight segmented negative sense single-stranded RNA molecules. Each
segment possesses segment-specific RNA packaging signals which are composed of both
the noncoding regions and short coding regions at both 5’ and 3’ ends. The eight
segmented RNAs encode 11 viral proteins, including RNA-dependent RNA polymerase
proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid; the
matrix membrane proteins (M1, M2); hemagglutinin (HA) and neuraminidase (NA),
both surface glycoproteins which project from the lipid-containing envelope; the
nonstructural protein (NS1), nuclear export protein (NEP, also termed NS2), the
proapoptotic factor PB1-F2. HA is critical for virus binding and entry to the cells, and is
the major neutralizing antibody target, whereas NA plays a role in progeny virus release
and is essential for virus propagation. Transcription and replication of the genome take
place in the nucleus and assembly occurs via budding on the plasma membrane. The
viruses can reassort genes during mixed infections.
Multiple swine influenza virus (SIV) subtypes continue to circulate in swine
populations despite available vaccines. Currently, H1N1, H3N2, and H1N2 are the
dominant subtypes that cause disease in the North American swine population. SIVs of
the subtype H3N2 were generated by reassortment between human, avian and classical
swine viruses, are undergoing rapid evolution and in general cause more severe disease
than classical H1N1 SIV. Current SIV vaccines do not provide cross-protection against
multiple antigenic SIV variants.
Thus, there remains a need for the development of effective strategies for the
treatment and prevention of swine influenza infection.
SUMMARY OF THE INVENTION
The present invention relates to recombinant, chimeric influenza viruses that
possess HAs from two or more subtypes of SIVs and methods of producing and using the
same. In preferred embodiments, all or a portion of the NA segment is absent from the
recombinant virus such that virus propagation is hindered. Because NA is essential for
virus propagation, the function of NA can be provided in culture by growing the virus in
the presence of sialidase. The recombinant virus that expresses more than one HA type
can be used in immunogenic compositions to stimulate an immune response against
influenza virus, and for treating and preventing influenza virus infection. Because HAs
from different subtypes of SIVs are present, compositions including the chimeric
influenza viruses can be used to provide broad coverage against a number of influenza
strains.
In particular, the inventors herein have found that a chimeric virus including both
H1 and H3, and retaining NA 3’ and 5’ viral RNA-specific packaging signals but lacking
the remainder of the NA segment, grows efficiently in culture and is attenuated in pigs as
no sialidase is present in swine. The NA packaging signals are largely retained for
efficient packaging. Such chimeric constructs can be used as effective and safe live,
attenuated vaccines.
Accordingly, in one embodiment, the invention is directed to a recombinant,
chimeric porcine influenza virus comprising more than one hemagglutinin (HA) segment
(segment 4) from more than one influenza subtype. In particular, the virus comprises
segments 1-5, 7 and 8 from a first influenza subtype and a second segment 4 from a
second influenza subtype. Further, all or a portion of the neuraminidase (NA) segment
(segment 6) of the first influenza subtype is missing to render an attenuated virus.
In certain embodiments, the second segment 4 comprises NA packaging
sequences from said first influenza subtype located 3’ and optionally 5’ to said second
segment 4. In additional embodiments, the NA packaging sequences comprise 3’ NA
packaging sequences from the 3’ NA UTR and the 3’ NA coding sequence and,
optionally 5’ NA packaging sequences from the 5’ NA UTR and the 5’ NA coding
sequence.
In further embodiments, the influenza virus described above is from an influenza
A virus. In certain embodiments, the influenza virus comprises an HA segment from an
H1N1 subtype and an HA segment from an H3N2 subtype. In certain embodiments, the
first influenza subtype is H1N1, such as A/swine/Saskatchewan/18789/02. In other
embodiments, the second influenza subtype is H3N2, such as A/Swine/Texas/4199-2/98.
In yet additional embodiments, the invention is directed to an attenuated,
recombinant, porcine influenza virus comprising segments 1-5, 7 and 8 from an H1N1
influenza subtype, and segment 4 from an H3N2 influenza subtype. Further, all or a
portion of segment 6 from the H1N1 influenza subtype is missing and the H3N2 segment
4 is flanked by NA packaging sequences from the H1N1 subtype. The packaging
sequences comprise 3’ NA packaging sequences from the 3’ NA UTR and the 3’ NA
coding sequence and 5’ NA packaging sequences from the 5’ NA UTR and the 5’ NA
coding sequence. In certain embodiments, the H1N1 subtype is
A/swine/Saskatchewan/18789/02 and the H3N2 subtype is A/Swine/Texas/4199-2/98.
In further embodiments, the invention is directed to a composition comprising
any one of the recombinant viruses described above, and a pharmaceutically acceptable
excipient. In certain embodiments, the composition further comprises an adjuvant. In
yet additional embodiments, the invention is directed to a method of eliciting an
immunological response in a vertebrate subject, comprising administering the
composition to the subject. In other embodiments, the invention is directed to a method
of treating or preventing an influenza infection in a vertebrate subject, comprising
administering to the subject a therapeutically effective amount of the composition. In
other embodiments, the invention is directed to a method of vaccinating a subject
against an influenza virus, comprising administering an effective amount of the
composition to the subject. In certain embodiments, the subject is a porcine subject.
In additional embodiments, the invention is directed to a recombinant construct
comprising: (a) a porcine influenza H3N2 subtype HA segment; and (b) porcine
influenza H1N1 subtype NA packaging sequences located 3’ and optionally 5’ to said
H3N2 HA segment. In certain embodiments, the H3N2 HA segment is flanked by H1N1
NA packaging sequences that comprise 3’ NA packaging sequences from the 3’ NA
UTR and the 3’ NA coding sequence and 5’ NA packaging sequences from the 5’ NA
UTR and the 5’ NA coding sequence. In additional embodiments the H1N1 subtype is
A/swine/Saskatchewan/18789/02 and the H3N2 subtype is A/Swine/Texas/4199-2/98.
In further embodiments, the invention is directed to a method of producing a
recombinant, chimeric influenza virus, comprising transfecting a host cell with (a)
individual plasmids comprising segments 1-5, 7 and 8 from an H1N1 influenza subtype;
and (b) a recombinant construct described above, and culturing the host cell under
conditions that result in the production of the recombinant, chimeric influenza virus.
In other embodiments, the invention is directed to a cell transformed with (a)
individual plasmids comprising segments 1-5, 7 and 8 from an H1N1 influenza subtype;
and (b) a recombinant construct as described above.
In further embodiments, the invention is directed to a method of producing a
composition comprising combining any of the recombinant, chimeric porcine influenza
viruses described above with a pharmaceutically acceptable excipient.
In other embodiments, the invention is directed to a method of producing an
influenza vaccine comprising: (a) propagating any one of the recombinant, chimeric
porcine influenza viruses described above; (b) purifying the virus; and (c) combining the
purified virus with a pharmaceutically acceptable excipient.
In yet additional embodiments, the invention is directed to a kit comprising one
or more containers of any one of the recombinant viruses described above, or the
compositions described above.
These and other embodiments of the subject invention will readily occur to those
of skill in the art in view of the disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1A and 1B depict various influenza segments for use in the present
invention. Figure 1A depicts the 8 segments of a wild-type Influenza A H1N1 SIV virus,
A/swine/Saskatchewan/18789/02 (termed “SK02” herein) and the 8 segments of the
recombinant, chimeric attenuated virus produced as described in the examples (termed
“SIV-606” herein). Figure 1B is a schematic representation of the segment termed “H3-
HA” in Figure 1A. The HA segment depicted in Figure 1B was derived from an H3N2
Influenza A virus, A/Swine/Texas/4199-2/98 (termed “Tx98” herein) and included the 3’
and 5’ NA packaging signals from SK02.
Figure 2 shows the growth curves of SIV-606 and SIV/SK02.
Figures 3A-3C show the body temperature of pigs infected with high and low
doses of SK02 (Figure 3A), Tx98 (Figure 3B) and SIV-606 (Figure 3C).
Figure 4 shows the lung virus titers of pigs infected with high and low doses of
SIV/SK02, SIV Tx98 and SIV-606.
Figures 5A and 5B (SEQ ID NOS:1 and 2) show the nucleotide sequence and
amino acid sequence, respectively, of HA from SIV SK02 (GenBank: AY619961.1).
Figures 6A and 6B (SEQ ID NOS:3 and 4) show the nucleotide sequence and
amino acid sequence, respectively, of NA from SIV SK02 (GenBank: AY619960.1).
Figures 7A-7C (SEQ ID NOS:5, 6 and 7) show the matrix nucleotide sequence
(Figure 7A) and the amino acid sequences of M2 (Figure 7B) and M1 (Figure 7C) from
SIV SK02 (GenBank: AY619959.1).
Figures 8A and 8B (SEQ ID NOS:8 and 9) show the nucleotide sequence and
amino acid sequence, respectively, of NP from SIV SK02 (GenBank: AY619958.1).
Figures 9A-9C (SEQ ID NOS:10, 11 and 12) show the nonstructural protein
nucleotide sequence (Figure 9A) and the amino acid sequences of NEP (Figure 9B) and
NS1 (Figure 9C) from SIV SK02 (GenBank: AY619957.1).
Figures 10A and 10B (SEQ ID NOS:13 and 14) show the nucleotide sequence
and amino acid sequence, respectively, of PA from SIV SK02 (GenBank: AY619956).
Figures 11A and 11B (SEQ ID NOS:15 and 16) show the nucleotide sequence
and amino acid sequence, respectively, of PB1 from SIV SK02 (GenBank:
AY619955.1).
Figures 12A and 12B (SEQ ID NOS:17 and 18) show the nucleotide sequence
and amino acid sequence, respectively, of PB2 from SIV SK02 (GenBank:
AY619954.1).
Figures 13A and 13B (SEQ ID NOS:19 and 20) show the nucleotide sequence
and amino acid sequence, respectively, of HA from SIV Tx98.
Figures 14A-14C show SIV/SK02-specific serum IgG titers (Figure 14A);
SIV/Tx98-specific serum IgG titers (Figure 14B); and H1N1 Halifax-specific serum IgG
titers (Figure 14C) in pigs vaccinated with SIV-606.
Figures 15A-15C show SIV/SK02-specific nasal IgA titers (Figure 15A);
SIV/Tx98-specific nasal IgA titers (Figure 15B); and H1N1 Halifax-specific nasal IgA
titers (Figure 15C) in pigs vaccinated with SIV-606.
Figures 16A-16C show SIV/SK02-specific BALF IgA titers (Figure 16A);
SIV/Tx98-specific BALF IgA titers (Figure 16B); and H1N1 Halifax-specific BALF IgA
titers (Figure 16C) in pigs vaccinated with SIV-606.
Figures 17A and 17B show rectal temperature in unvaccinated control pigs and
SIV-606 vaccinated pigs challenged with SIV/SK02 (Figure 17A) and challenged with
SIV/Tx98 (Figure 17B).
Figures 18A and 18B show the percentage of lung lesions (Figure 18A) and lung
viral load (Figure 18B) in unvaccinated SIV/SK02 and SIV/Tx98 pigs, as well as in pigs
vaccinated with SIV-606.
Figures 19A-19E show histopathological lesions in unvaccinated, unchallenged
pigs (Figure 19A); MEM vaccinated and challenged pigs (Figure 19B); SIV-606
vaccinated and SIV/SK02 challenged pigs (Figure 19C); MEM vaccinated and SIV/Tx98
challenged pigs (Figure 19D); and SIV-606 vaccinated and SIV/Tx98 challenged pigs
(Figure 19E).
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise indicated,
conventional methods of virology, chemistry, biochemistry, recombinant DNA
techniques and immunology, within the skill of the art. Such techniques are explained
fully in the literature. See, e.g., Fundamental Virology, Current Edition, vol. I & II (B.N.
Fields and D.M. Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D.M.
Weir and C.C. Blackwell eds., Blackwell Scientific Publications); T.E. Creighton,
Proteins: Structures and Molecular Properties (W.H. Freeman and Company); A.L.
Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook, et al.,
Molecular Cloning: A Laboratory Manual (current edition); Methods In Enzymology (S.
Colowick and N. Kaplan eds., Academic Press, Inc.).
The following amino acid abbreviations are used throughout the text:
Alanine: Ala (A) Arginine: Arg (R)
Asparagine: Asn (N) Aspartic acid: Asp (D)
Cysteine: Cys (C) Glutamine: Gln (Q)
Glutamic acid: Glu (E) Glycine: Gly (G)
Histidine: His (H) Isoleucine: Ile (I)
Leucine: Leu (L) Lysine: Lys (K)
Methionine: Met (M) Phenylalanine: Phe (F)
Proline: Pro (P) Serine: Ser (S)
Threonine: Thr (T) Tryptophan: Trp (W)
Tyrosine: Tyr (Y) Valine: Val (V)
1. DEFINITIONS
In describing the present invention, the following terms will be employed, and
are intended to be defined as indicated below.
It must be noted that, as used in this specification and the appended claims, the
singular forms “a”, “an” and “the” include plural referents unless the content clearly
dictates otherwise. Thus, for example, reference to “an influenza A virus” includes a
mixture of two or more such viruses, and the like.
As used herein, the term “influenza virus” refers to members of the
orthomyxoviridae family of enveloped viruses with a segmented antisense RNA genome
(Knipe and Howley (eds.) Fields Virology, 4th edition, Lippincott Williams and Wilkins,
Philadelphia, Pa., 2001). The term influenza virus may include any strain of influenza
virus, such as influenza A, B, or C, which is capable of causing disease in an animal or
human subject. In particular, the term encompasses any subtype of influenza A virus
selected from H1-H15 and N1-N9, such as but not limited to H1N1, H1N2, H3N2,
H3N1, H9N2 and H5N1, or any combination of H’s and N’s. A large number of
influenza isolates have been partially or completely sequenced. See, e.g., the Influenza
Sequence Database (ISD) (website at flu.lanl.gov; described by Macken et al., "The
value of a database in surveillance and vaccine selection." in Options for the Control of
Influenza IV. A.D.M.E. Osterhaus, N. Cox & A.W. Hampson (Eds.) Amsterdam:
Elsevier Science, 2001, 103-106) and the GenBank database, particularly the Influenza
Virus Resource (website at ncbi.nlm.nih.gov/genomes/FLU/FLU.html). The ISD and
GenBank databases contain complete sequences for influenza A, B and C genome
segments.
The term “derived from” is used herein to identify the original source of a
molecule but is not meant to limit the method by which the molecule is made which can
be, for example, by chemical synthesis or recombinant means.
An influenza virus molecule is a molecule derived from an influenza virus,
including, without limitation, polypeptide, protein, polynucleotide, oligonucleotide, and
nucleic acid molecules, as defined herein, from any of the various isolates of influenza
subtypes A, B, or C. The molecule need not be physically derived from the particular
isolate in question, but may be synthetically or recombinantly produced.
Nucleic acid and polypeptide sequences for a number of influenza virus isolates
are known. Representative influenza sequences are presented in Figures 5-13 herein.
Additional representative sequences, including additional sequences for the 8 influenza
segments, including those segments coding for hemagglutinin (HA), neuraminidase
(NA), polymerase acidic protein (PA), polymerase basic proteins 1 and 2 (PB1 and PB2),
matrix membrane proteins 1 and 2 (M1 and M2), nucleoprotein (NP), and nonstructural
proteins 1 and 2 (NS1 and NEP, also termed NS2) from influenza isolates found in
various species are listed in the National Center for Biotechnology Information (NCBI)
database and the Influenza Research Database found at fludb.org. See also Ferguson et
al. (2003) Nature 422: 428-433; Lin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 9654-
9658; Nguyen et al. (2005) J. Virol. 79:4201-4212; Ha et al. (2002) EMBO J. 21:865-
875; and Chan et al. (2004) J. Microbiol. Immunol. Infect. 37:135-144; for sequence
comparisons and a discussion of genetic diversity and phylogenetic analysis of influenza
virus.
As used herein, the term “swine influenza virus” refers to a type A or type C
influenza virus from the family orthomyxovirus that causes swine influenza. While
orthomyxovirus has three groups: type A, type B and type C, only type A and type C
influenza viruses infect pigs. Subtypes of swine influenza virus include H1N1, H1N2,
H3N2, H3N1, H9N2 and H5N1. In certain embodiments, a swine influenza virus is an
influenza virus that has been isolated from swine. For purposes of the present invention,
a swine influenza virus is either a wild-type swine influenza virus or a recombinant,
chimeric influenza virus derived from a wild-type swine influenza virus.
As used herein, the phrase “wild-type swine influenza virus” refers to the types of
a swine virus that are prevalent, circulating naturally and producing typical outbreaks of
disease. Examples of wild-type swine influenza viruses include, but are not limited to,
A/swine/Saskatchewan/18789/02, A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99,
A/Swine/Cote d'Armor/3633/84, A/Swine/Cote d'Armor/3633/84,
A/Swine/England/195852/92, A/Swine/Finistere/2899/82, A/Swine/Hong Kong/10/98,
A/Swine/Hong Kong/9/98, A/Swine/Hong Kong/81/78, A/Swine/Illinois/100084/01,
A/Swine/Illinois/100085A/01, A/Swine/Illinois/21587/99, A/Swine/Indiana/1726/88,
A/Swine/Indiana/9K035/99, A/Swine/Indiana/P 12439/00, A/Swine/Iowa/30,
A/Swine/Iowa/15/30, A/Swine/Iowa/533/99, A/Swine/Iowa/569/99,
A/Swine/Iowa/3421/90, A/Swine/Iowa/8548-1/98, A/Swine/Iowa/930/01,
A/Swine/Iowa/17672/88, A/Swine/Italy/1513-1/98, A/Swine/Italy/1523/98,
A/Swine/Korea/CY02/02, A/Swine/Minnesota/55551/00, A/Swine/Minnesota/593/99,
A/Swine/Minnesota/9088-2/98, A/Swine/Nebraska/1/92, A/Swine/Nebraska/209/98,
A/Swine/Netherlands/12/85, A/Swine/North Carolina/16497/99, A/Swine/North
Carolina/35922/98, A/Swine/North Carolina/93523/01, A/Swine/North
Carolina/98225/01, A/Swine/Oedenrode/7C/96, A/Swine/Ohio/891/01,
A/Swine/Oklahoma/18717/99, A/Swine/Oklahoma/18089/99, A/Swine/Ontario/01911-
1/99, A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97, A/Swine/Ontario/97,
A/Swine/Quebec/192/81, A/Swine/Quebec/192/91, A/Swine/Quebec/5393/91,
A/Swine/Taiwan/7310/70, A/Swine/Tennessee/24/77, A/Swine/Texas/4199-2/98,
A/Swine/Wisconsin/125/97, A/Swine/Wisconsin/136/97, A/Swine/Wisconsin/163/97,
A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97, A/Swine/Wisconsin/168/97,
A/Swine/Wisconsin/235/97, A/Swine/Wisconsin/238/97, A/Swine/Wisconsin/457/98,
A/Swine/Wisconsin/458/98, A/Swine/Wisconsin/464/98 and
A/Swine/Wisconsin/14094/99.
The term “HA gene” refers to the gene which encodes the hemagglutinin (HA)
surface glycoprotein which projects from the lipid-containing envelope in influenza. HA
is one of the molecules encoded by the segmented genome of influenza A and other
viruses. A “swine influenza virus HA gene” is an HA gene isolated from a swine
influenza virus, such as from any of the strains described above. The polynucleotide and
amino acid sequences of representative swine HA genes can be found in public sequence
databases such as GenBank. For example, HA genes from H1N1 and include, but are
not limited to, GenBank Accession Nos. AY619961.1 (see Figures 5A and 5B);
GQ457549.1; GQ457548.1; GQ457547.1; CY091769.1; CY091745.1; CY091737.1;
CY091729.1; GU721143.3; JF820285.1; JF820277.1; JF707784.1; CY087136.1;
CY087104.1; CY087096.1; CY087080.1; CY087072.1; CY087064.1; CY087056.1;
CY087048.1; CY086863.1; CY086839.1; CY086353.1; CY086006.1; CY085990.1;
CY085982.1; CY085974.1; CY085966.1; CY085958.1; CY085950.1; CY085942.1;
CY085934.1; CY085926.1; CY085918.1; CY085910.1; CY085902.1; CY085894.1;
CY085886.1; CY085878.1; CY085870.1; CY085854.1; CY085846.1; CY085838.1;
CY085830.1; CY085822.1; CY085814.1; CY085806.1; CY085798.1; CY085790.1;
CY085782.1; CY085774.1; CY085766.1; CY085758.1; CY085742.1; CY085726.1;
CY085718.1; CY085710.1; CY085702.1; CY085694.1; CY085686.1; CY085670.1;
JF833344.1; JF833343.1; JF833341.1; JF833339.1; JF833338.1; JF833337.1;
JF833335.1; JF916682.1; JF812292.1; JF812291.1; JF812290.1; JF812287.1;
JF812284.1; JF812281.1; JF812280.1; JF812279.1; JF812278.1; JF812273.1;
JF812272.1; JF812271.1; AF091317.1; AF091315.1; AF091314.1.
HA genes from H3N2 and include, but are not limited to, the sequence shown in
Figures 13A and 13B; as well as GenBank Accession Nos. AY377927.2; CY092324.1;
AF153233.1; JN105973.1; HQ315643.1; FJ519977.1; FJ519976.1; FJ519975.1;
FJ519974.1; FJ519973.1; FJ519972.1; FJ519971.1; GU937743.1; JF833345.1;
JF833340.1; JF833336.1; JF833334.1; JF812293.1; JF812289.1; JF812277.1;
JF812276.1; JF812275.1; JF812274.1; CY045575.1; CY045567.1; CY045559.1;
CY045551.1; HQ825243.1; HQ825235.1; HQ825229.1; HQ825226.1; HQ825223.1;
HQ825218.1; HQ825210.1; HQ825210.1; HQ825198.1; HQ825190.1; HQ825182.1;
HQ825174.1; HQ825166.1; JF312065.1; JF312064.1; CY086920.1; JF312073.1;
JF312072.1; JF312071.1; JF316643.1; JF263536.1; JF263535.1; HQ734204.1;
HQ734201.1; HQ734198.1; HQ734195.1; HQ734192.1; HQ734189.1; HQ734186.1;
CY077942.1; CY077934.1.
The term “NA gene” refers to the gene which encodes the neuraminidase (NA)
surface glycoprotein which projects from the lipid-containing envelope in influenza. NA
is one of the molecules encoded by the segmented genome of influenza A and other
viruses. A “swine influenza virus NA gene” is an NA gene isolated from a swine
influenza virus, such as from any of the strains described above. The polynucleotide and
amino acid sequences of representative swine NA genes can be found in public sequence
databases such as GenBank. For example, NA genes from H1N1 and include, but are
not limited to, AY619960.1 (see Figures 6A and 6B); JF833356.1.; JF833355.1;
JF833353.1; JF833351.1; JF833350.1; JF833349.1; JF833355.1; JF833347.1;
JF812315.1; JF812314.1; JF812313.1; JF812310.1; JF812307.1; JF812304.1;
JF812303.1; JF812302.1; JF812301.1; JF812294.1; FJ791299.1; FJ791298.1;
FJ791297.1; FJ791296.1; FJ791295.1; FJ791294.1; FJ791293.1; FJ791292.1;
FJ791291.1; FJ791290.1; FJ791289.1; FJ791288.1; FJ791287.1.
The term “NA packaging signal” refers to the 3’ and 5’ viral RNA-specific
packaging signals for NA that provide for efficient incorporation of viral RNA into viral
particles. The packaging signals are present in the 5’ and 3’ untranslated regions (UTRs)
and extend into the coding region of the NA segment. Preferably, the NA packaging
signals used in the production of the recombinant, chimeric viruses will include only so
much of the NA region sufficient for packaging and will not include the entire NA
coding sequence. NA packaging signals are discussed in greater detail below.
As used herein, the phrase “multiplicity of infection” or “MOI” is the average
number of virus per infected cell. The MOI is determined by dividing the number of
virus added (ml added x PFU) by the number of cells added (ml added x cells/ml).
As used herein, the term “attenuated” means that an influenza virus variant, such
as a recombinant, chimeric virus described herein, exhibits a measurable reduction in
replication efficiency relative to wild-type influenza virus. The replication efficiency of
an influenza virus may be determined, for example, by measuring plaque size in MDCK
cells, by measuring virus titers over multiple growth cycles, or by isolating virus from
infected lung tissue and measuring titers.
The terms “polypeptide” and “protein” refer to a polymer of amino acid residues
and are not limited to a minimum length of the product. Thus, peptides, oligopeptides,
dimers, multimers, and the like, are included within the definition. Both full-length
proteins and fragments thereof are encompassed by the definition. The terms also
include postexpression modifications of the polypeptide, for example, glycosylation,
acetylation, phosphorylation and the like. Furthermore, for purposes of the present
invention, a “polypeptide” refers to a protein which includes modifications, such as
deletions, additions and substitutions, to the native sequence, so long as the protein
maintains the desired activity. These modifications may be deliberate, as through
site-directed mutagenesis, or may be accidental, such as through mutations of hosts
which produce the proteins or errors due to PCR amplification.
“Substantially purified” generally refers to isolation of a substance (recombinant
virus, compound, polynucleotide, protein, polypeptide, polypeptide composition) such
that the substance comprises the majority percent of the sample in which it resides.
Typically in a sample, a substantially purified component comprises 50%, preferably
80%-85%, more preferably 90-95% of the sample. Techniques for purifying molecules
of interest are well-known in the art and include, for example, ion-exchange
chromatography, affinity chromatography and sedimentation according to density.
By “isolated” is meant, when referring to a polypeptide, that the indicated
molecule is separate and discrete from the whole organism with which the molecule is
found in nature or is present in the substantial absence of other biological
macro-molecules of the same type. The term “isolated” with respect to a polynucleotide
is a nucleic acid molecule devoid, in whole or part, of sequences normally associated
with it in nature; or a sequence, as it exists in nature, but having heterologous sequences
in association therewith; or a molecule disassociated from the chromosome.
“Homology” refers to the percent identity between two polynucleotide or two
polypeptide moieties. Two nucleic acid, or two polypeptide sequences are “substantially
homologous” to each other when the sequences exhibit at least about 50% sequence
identity, preferably at least about 75% sequence identity, more preferably at least about
80%-85% sequence identity, more preferably at least about 90% sequence identity, and
most preferably at least about 95%-98% sequence identity over a defined length of the
molecules. As used herein, substantially homologous also refers to sequences showing
complete identity to the specified sequence.
In general, “identity” refers to an exact nucleotide-to-nucleotide or amino
acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences,
respectively. Percent identity can be determined by a direct comparison of the sequence
information between two molecules by aligning the sequences, counting the exact
number of matches between the two aligned sequences, dividing by the length of the
shorter sequence, and multiplying the result by 100. Readily available computer
programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of
Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353-358, National
biomedical Research Foundation, Washington, DC, which adapts the local homology
algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 for peptide
analysis. Programs for determining nucleotide sequence identity are available in the
Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer
Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which
also rely on the Smith and Waterman algorithm. These programs are readily utilized
with the default parameters recommended by the manufacturer and described in the
Wisconsin Sequence Analysis Package referred to above. For example, percent identity
of a particular nucleotide sequence to a reference sequence can be determined using the
homology algorithm of Smith and Waterman with a default scoring table and a gap
penalty of six nucleotide positions.
Another method of establishing percent identity in the context of the present
invention is to use the MPSRCH package of programs copyrighted by the University of
Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by
IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the
Smith-Waterman algorithm can be employed where default parameters are used for the
scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a
gap of six). From the data generated the “Match” value reflects "sequence identity."
Other suitable programs for calculating the percent identity or similarity between
sequences are generally known in the art, for example, another alignment program is
BLAST, used with default parameters. For example, BLASTN and BLASTP can be
used using the following default parameters: genetic code = standard; filter = none;
strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50
sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL +
DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details
of these programs are readily available.
Alternatively, homology can be determined by hybridization of polynucleotides
under conditions which form stable duplexes between homologous regions, followed by
digestion with single-stranded-specific nuclease(s), and size determination of the
digested fragments. DNA sequences that are substantially homologous can be identified
in a Southern hybridization experiment under, for example, stringent conditions, as
defined for that particular system. Defining appropriate hybridization conditions is
within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra;
Nucleic Acid Hybridization, supra.
The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid
molecule” are used herein to include a polymeric form of nucleotides of any length,
either ribonucleotides or deoxyribonucleotides. This term refers only to the primary
structure of the molecule. Thus, the term includes triple-, double- and single-stranded
DNA, as well as triple-, double- and single-stranded RNA. It also includes
modifications, such as by methylation and/or by capping, and unmodified forms of the
polynucleotide. More particularly, the terms “polynucleotide,” “oligonucleotide,”
“nucleic acid” and “nucleic acid molecule” include polydeoxyribonucleotides
(containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type
of polynucleotide which is an N– or C-glycoside of a purine or pyrimidine base, and
other polymers containing nonnucleotidic backbones, for example, polyamide (e.g.,
peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the
Anti-Virals, Inc., Corvallis, Oregon, as Neugene) polymers, and other synthetic
sequence-specific nucleic acid polymers providing that the polymers contain nucleobases
in a configuration which allows for base pairing and base stacking, such as is found in
DNA and RNA. There is no intended distinction in length between the terms
“polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and
these terms will be used interchangeably. Thus, these terms include, for example,
3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates,
2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and
single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or
RNA, and also include known types of modifications, for example, labels which are
known in the art, methylation, “caps,” substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g.,
aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant
moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal
peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.),
those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.),
those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic
acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide. In
particular, DNA is deoxyribonucleic acid.
A polynucleotide “derived from” a designated sequence refers to a polynucleotide
sequence which comprises a contiguous sequence of approximately at least about 6
nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12
nucleotides, and even more preferably at least about 15-20 nucleotides corresponding,
i.e., identical or complementary to, a region of the designated nucleotide sequence. The
derived polynucleotide will not necessarily be derived physically from the nucleotide
sequence of interest, but may be generated in any manner, including, but not limited to,
chemical synthesis, replication, reverse transcription or transcription, which is based on
the information provided by the sequence of bases in the region(s) from which the
polynucleotide is derived. As such, it may represent either a sense or an antisense
orientation of the original polynucleotide.
“Recombinant” as used herein to describe a nucleic acid molecule means a
polynucleotide of genomic, RNA, cDNA, viral, semisynthetic, or synthetic origin which,
by virtue of its origin or manipulation is not associated with all or a portion of the
polynucleotide with which it is associated in nature. The term “recombinant” as used
with respect to a virus, means a virus produced by manipulation of the viral genome.
“Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and
other such terms denoting microorganisms or higher eukaryotic cell lines cultured as
unicellular entities refer to cells which can be, or have been, used as recipients for
recombinant viruses and vectors or other transferred nucleic acid, and include the
original progeny of the original cell which has been transfected.
A “coding sequence” or a sequence which “encodes” a selected polypeptide, is a
nucleic acid molecule which is transcribed and translated into a polypeptide in vivo when
placed under the control of appropriate regulatory sequences (or “control elements”).
The boundaries of the coding sequence can be determined by a start codon at the 5'
(amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding
sequence can include, but is not limited to, RNA or cDNA from viral, procaryotic or
eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even
synthetic DNA sequences. A transcription termination sequence may be located 3' to the
coding sequence.
Typical “control elements” include, but are not limited to, transcription
promoters, transcription enhancer elements, transcription termination signals,
polyadenylation sequences (located 3' to the translation stop codon), sequences for
optimization of initiation of translation (located 5’ to the coding sequence), and
translation termination sequences.
“Operably linked” refers to an arrangement of elements wherein the components so
described are configured so as to perform their usual function. Thus, a given promoter
operably linked to a coding sequence is capable of effecting the expression of the coding
sequence when the proper enzymes are present. The promoter need not be contiguous
with the coding sequence, so long as it functions to direct the expression thereof. Thus,
for example, intervening untranslated yet transcribed sequences can be present between
the promoter sequence and the coding sequence and the promoter sequence can still be
considered “operably linked” to the coding sequence.
“Encoded by” refers to a nucleic acid sequence which codes for a polypeptide
sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid
sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and
even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the
nucleic acid sequence.
“Expression cassette” or “expression construct” refers to an assembly which is
capable of directing the expression of the sequence(s) or gene(s) of interest. An
expression cassette generally includes control elements, as described above, such as a
promoter which is operably linked to (so as to direct transcription of) the sequence(s) or
gene(s) of interest, and often includes a polyadenylation sequence as well. An
expression cassette may be contained within a plasmid construct. In addition to the
components of the expression cassette, the plasmid construct may also include, one or
more selectable markers, a signal which allows the plasmid construct to exist as
single-stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning
site, and a “mammalian” origin of replication (e.g., a SV40 or adenovirus origin of
replication).
The term “transfection” is used to refer to the uptake of foreign nucleic acid by a
cell. A cell has been "transfected" when exogenous nucleic acid has been introduced
inside the cell membrane. A number of transfection techniques are generally known in
the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989)
Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York,
Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981)
Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA
moieties into suitable host cells. The term refers to both stable and transient uptake of
the genetic material, and includes uptake of peptide- or antibody-linked nucleic acids.
A "vector" is capable of transferring nucleic acid sequences to target cells (e.g.,
viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector
construct," "expression vector, " and "gene transfer vector," mean any nucleic acid
construct capable of directing the expression of a nucleic acid of interest and which can
transfer nucleic acid sequences to target cells. Thus, the term includes cloning and
expression vehicles, as well as viral vectors.
An “immunological response” to an antigen or composition is the development in
a subject of a humoral and/or a cellular immune response to an antigen present in the
composition of interest. For purposes of the present invention, a “humoral immune
response” refers to an immune response mediated by antibody molecules, while a
“cellular immune response” is one mediated by T-lymphocytes and/or other white blood
cells. One important aspect of cellular immunity involves an antigen-specific response
by cytolytic T-cells (“CTL”s). CTLs have specificity for peptide antigens that are
presented in association with proteins encoded by the major histocompatibility complex
(MHC) and expressed on the surfaces of cells. CTLs help induce and promote the
destruction of intracellular microbes, or the lysis of cells infected with such microbes.
Another aspect of cellular immunity involves an antigen-specific response by helper T-
cells. Helper T-cells act to help stimulate the function, and focus the activity of,
nonspecific effector cells against cells displaying peptide antigens in association with
MHC molecules on their surface. A “cellular immune response” also refers to the
production of cytokines, chemokines and other such molecules produced by activated T-
cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-
cells.
A composition or vaccine that elicits a cellular immune response may serve to
sensitize a vertebrate subject by the presentation of antigen in association with MHC
molecules at the cell surface. The cell-mediated immune response is directed at, or near,
cells presenting antigen at their surface. In addition, antigen-specific T-lymphocytes can
be generated to allow for the future protection of an immunized host.
The ability of a particular antigen to stimulate a cell-mediated immunological
response may be determined by a number of assays, such as by lymphoproliferation
(lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-
lymphocytes specific for the antigen in a sensitized subject. Such assays are well known
in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al., Eur.
J. Immunol. (1994) 24:2369-2376. Recent methods of measuring cell-mediated immune
response include measurement of intracellular cytokines or cytokine secretion by T-cell
populations, or by measurement of epitope specific T-cells (e.g., by the tetramer
technique)(reviewed by McMichael, A.J., and O’Callaghan, C.A., J. Exp. Med. (1998)
187:1367-1371; Mcheyzer-Williams, M.G., et al, Immunol. Rev. (1996) 150:5-21;
Lalvani, A., et al, J. Exp. Med. (1997) 186:859-865).
Thus, an immunological response as used herein may be one that stimulates the
production of antibodies (e.g., neutralizing antibodies that block pathogens such as
viruses entering cells and replicating by binding to toxins and pathogens, typically
protecting cells from infection and destruction). The antigen of interest may also elicit
production of CTLs. Hence, an immunological response may include one or more of the
following effects: the production of antibodies by B-cells; and/or the activation of
suppressor T-cells and/or memory/effector T-cells directed specifically to an antigen or
antigens present in the composition or vaccine of interest. These responses may serve to
neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell
cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be
determined using standard immunoassays and neutralization assays, well known in the
art. (See, e.g., Montefiori et al. J. Clin Microbiol. (1988) 26:231-235; Dreyer et al.,
AIDS Res Hum Retroviruses (1999) 15:1563-1571). The innate immune system of
mammals also recognizes and responds to molecular features of pathogenic organisms
via activation of Toll-like receptors and similar receptor molecules on immune cells.
Upon activation of the innate immune system, various non-adaptive immune response
cells. are activated to, e.g., produce various cytokines, lymphokines and chemokines.
Cells activated by an innate immune response include immature and mature Dendritic
cells of the monocyte and plamsacytoid lineage (MDC, PDC), as well as gamma, delta,
alpha and beta T cells and B cells and the like. Thus, the present invention also
contemplates an immune response wherein the immune response involves both an innate
and adaptive response.
An “immunogenic composition” is a composition that comprises an antigenic
molecule where administration of the composition to a subject results in the development
in the subject of an immunological response as defined above.
An “antigen” refers to a molecule, such as a protein, polypeptide, or fragment
thereof, or an attenuated virus, containing one or more epitopes (either linear,
conformational or both) that will stimulate a host's immune-system to make an
immunological response, as defined above. The term is used interchangeably with the
term "immunogen." The term “antigen” denotes both subunit antigens, (i.e., antigens
which are separate and discrete from a whole organism with which the antigen is
associated in nature), as well as, killed, attenuated or inactivated bacteria, viruses, fungi,
parasites or other microbes. Similarly, an oligonucleotide or polynucleotide which
expresses an antigen or antigenic determinant in vivo, such as in gene therapy and
nucleic acid immunization applications, is also included in the definition of antigen
herein.
By “vertebrate subject” is meant any member of the subphylum chordata,
including, without limitation, humans and other primates, including non-human primates
such as chimpanzees and other apes and monkey species; farm animals such as cattle,
sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory
animals including rodents such as mice, rats and guinea pigs; birds, including domestic,
wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese,
and the like. The term does not denote a particular age. Thus, both adult and newborn
individuals are intended to be covered.
By “therapeutically effective amount” in the context of the immunogenic
compositions is meant an amount of an immunogen, e.g., a recombinant, chimeric
influenza virus, which will induce an immunological response, either for antibody
production or for treatment or prevention of influenza virus infection. Such a response
will generally result in the development in the subject of an antibody-mediated and/or a
secretory or cellular immune response to the composition. Usually, such a response
includes but is not limited to one or more of the following effects; the production of
antibodies from any of the immunological classes, such as immunoglobulins A, D, E, G
or M; the proliferation of B and T lymphocytes; the provision of activation, growth and
differentiation signals to immunological cells; expansion of helper T cell, suppressor T
cell, and/or cytotoxic T cell and/or γδT cell populations.
“Parenteral administration” refers to introduction into the body outside the
digestive tract, such as by subcutaneous, intramuscular, intradermal or intravenous
administration. This is to be contrasted with delivery to a mucosal surface, such as oral,
nasal, vaginal or rectal. “Mucosal administration” refers to introduction into the body
via any mucosal surface, such as intragastrically, pulmonarily, transdermally,
intestinally, ocularly, intranasally, orally, vaginally, rectally, intratracheally or the like.
As used herein, “treatment” refers to any of (i) the prevention of infection or
reinfection, as in a traditional vaccine, (ii) the reduction or elimination of symptoms, and
(iii) the substantial or complete elimination of influenza virus from an infected
individual. Treatment may be effected prophylactically (prior to infection) or
therapeutically (following infection).
2. MODES OF CARRYING OUT THE INVENTION
Before describing the present invention in detail, it is to be understood that this
invention is not limited to particular formulations or process parameters as such may, of
course, vary. It is also to be understood that the terminology used herein is for the
purpose of describing particular embodiments of the invention only, and is not intended
to be limiting.
Although a number of methods and materials similar or equivalent to those
described herein can be used in the practice of the present invention, the preferred
materials and methods are described herein.
The present invention provides recombinant, chimeric swine influenza
viruses which are attenuated with an impaired ability to replicate in vivo, methods
for producing such attenuated swine influenza viruses, and the use of such viruses
in vaccine and pharmaceutical formulations. Such viruses are capable of
generating an immune response and creating immunity but either do not cause
illness or cause fewer and/or less severe symptoms, i.e., the viruses have decreased
virulence. Therefore, they are ideal candidates for live virus vaccines. Moreover,
because HAs from different subtypes of SIVs are present, compositions including
the chimeric influenza viruses can be used to provide broad coverage against a
number of influenza strains.
In particular, the invention pertains to recombinant, chimera influenza
viruses that include HA segments from more than one influenza subtype and
include a deletion of all or part of the NA segment, immunogenic compositions
comprising the viruses, as well as methods of stimulating an immune response
against influenza virus, and methods of interfering with influenza virus replication.
In order to further an understanding of the invention, a more detailed
discussion is provided below regarding the production of recombinant, chimeric
influenza viruses and methods of using the same in compositions in the treatment
and/or prevention of influenza virus infection.
A. Recombinant, Chimeric Influenza Viruses
Wild-type swine influenza viruses typically include an 8 segmented genome with
the segments designated as follows:
SEGMENT GENE PRODUCT NAME
1 PB2 (Polymerase (basic) protein 2)
2 PB1 (Polymerase (basic) protein 1)
3 PA (Polymerase (acidic) protein)
4 HA (Hemagglutinin)
NP (Nucleoprotein)
6 NA (Neuraminidase)
7 M1 (Matrix protein 1); M2 (Matrix protein 2)
8 NS1 (Non-structural protein 1); NEP, also termed NS2 (Non-
structural protein 2)
The recombinant, chimeric influenza viruses described herein include two or
more HA segments (segment 4) from two or more subtypes of influenza viruses. The
recombinant influenza viruses can include HAs from any subtype of influenza virus and
preferably from influenza A virus, selected from H1-H15 and N1-N9, such as but not
limited to H1N2, H1N1, H3N2, H3N1, H9N2 and H5N1or any combination of H’s and
N’s. Particularly preferred are HA segments from viruses that infect pigs. The
polynucleotide and amino acid sequences of representative swine HA genes can be found
in public sequence databases such as GenBank. For example, HA genes from H1N1 and
include, but are not limited to, GenBank Accession Nos. AY619961.1 (see Figures 5A
and 5B); GQ457549.1; GQ457548.1; GQ457547.1; CY091769.1; CY091745.1;
CY091737.1; CY091729.1; GU721143.3; JF820285.1; JF820277.1; JF707784.1;
CY087136.1; CY087104.1; CY087096.1; CY087080.1; CY087072.1; CY087064.1;
CY087056.1; CY087048.1; CY086863.1; CY086839.1; CY086353.1; CY086006.1;
CY085990.1; CY085982.1; CY085974.1; CY085966.1; CY085958.1; CY085950.1;
CY085942.1; CY085934.1; CY085926.1; CY085918.1; CY085910.1; CY085902.1;
CY085894.1; CY085886.1; CY085878.1; CY085870.1; CY085854.1; CY085846.1;
CY085838.1; CY085830.1; CY085822.1; CY085814.1; CY085806.1; CY085798.1;
CY085790.1; CY085782.1; CY085774.1; CY085766.1; CY085758.1; CY085742.1;
CY085726.1; CY085718.1; CY085710.1; CY085702.1; CY085694.1; CY085686.1;
CY085670.1; JF833344.1; JF833343.1; JF833341.1; JF833339.1; JF833338.1;
JF833337.1; JF833335.1; JF916682.1; JF812292.1; JF812291.1; JF812290.1;
JF812287.1; JF812284.1; JF812281.1; JF812280.1; JF812279.1; JF812278.1;
JF812273.1; JF812272.1; JF812271.1; AF091317.1; AF091315.1; AF091314.1.
HA genes from H3N2 and include, but are not limited to, GenBank Accession
Nos. AF153233.1 (see Figures 13A and 13B); AY377927.2; CY092324.1; JN105973.1;
HQ315643.1; FJ519977.1; FJ519976.1; FJ519975.1; FJ519974.1; FJ519973.1;
FJ519972.1; FJ519971.1; GU937743.1; JF833345.1; JF833340.1; JF833336.1;
JF833334.1; JF812293.1; JF812289.1; JF812277.1; JF812276.1; JF812275.1;
JF812274.1; CY045575.1; CY045567.1; CY045559.1; CY045551.1; HQ825243.1;
HQ825235.1; HQ825229.1; HQ825226.1; HQ825223.1; HQ825218.1; HQ825210.1;
HQ825210.1; HQ825198.1; HQ825190.1; HQ825182.1; HQ825174.1; HQ825166.1;
JF312065.1; JF312064.1; CY086920.1; JF312073.1; JF312072.1; JF312071.1;
JF316643.1; JF263536.1; JF263535.1; HQ734204.1; HQ734201.1; HQ734198.1;
HQ734195.1; HQ734192.1; HQ734189.1; HQ734186.1; CY077942.1; CY077934.1.
Any of the above HAs or other readily available HA sequences can be used with
the subject invention.
Additionally, the recombinant, chimeric influenza viruses typically include a
mutation in the NA genomic segment (segment 6) coding for neuraminidase such that
replication of the virus is impaired. Mutations can include deletions, inversions,
insertions or substitutions that impair replication of the virus. In certain embodiments,
the virus variant comprises a deletion of all or part of the NA segment such that virus
propagation is hindered. Because NA is essential for virus propagation, the function of
NA can be provided in culture by growing the virus in the presence of sialidase.
Preferably, NA packaging sequences at the 3’ and optionally the 5’ untranslated regions
(UTRs) flanking the NA sequence and extending into the coding sequence are retained in
the recombinant viruses.
In particular, specific cis-acting packaging signals exist in 3’ and 5’ (UTRs) that
extend into the coding regions of most if not all segments, including the NA segment,
which is responsible for viral release from infected cells by removing sialic acids from
cellular glycoconjugates and viral glycoproteins. Each viral RNA consists
predominantly of coding sequences (in antisense orientation), flanked at both ends by
UTRs that range from 19 to 58 bases long. Within these UTRs, the distal 12 and 13
noncoding bases that form the extreme 3’ and 5’ termini, respectively, of every segment
are highly conserved among viral strains and among the eight segments themselves.
These distal conserved sequences are partially complementary to each other and can
anneal to form a bulged duplex structure that is essential for transcription and replication
of the segment. The UTRs harbor cis-acting signals that contribute to RNA packaging,
since the attachment of authentic UTRs onto a heterologous RNA can enable it to be
packaged into, and transduced by, influenza virus particles. Optimal packaging of at
least some segments, such as NA, HA and NS requires not only both UTRs but also short
portions of the coding region.
Deletion analysis of reporter constructs indicates that the minimal sequences
needed for efficient packaging extend beyond each UTR to include 9 to 80 bases of
adjacent coding sequence at either end of the segment (Fujii et al., J. Virol. (2005)
79:3766–3774; Fujii et al., Proc. Natl. Acad. Sci. USA (2003) 100:2002–2007;
Watanabe et al., J. Virol. (2003) 77:10575–10583). Sequences at the 3’ end of the
coding region appear to exert a greater quantitative effect than those at the 5’ end. These
regions are therefore useful for packaging and maintaining wild-type NA RNA as well as
mutant NA RNAs, e.g., RNAs with internal deletions and/or insertions. Accordingly, the
recombinant, chimeric viruses of the invention will include at least packaging signals
from the 3’ UTR and a portion of the 3’ NA coding region, and preferably will include
packaging signals from both the 3’ and 5’ UTRs and 3’ and 5’ portions of the NA
sequence.
Methods for locating packaging signals are known. In particular, Gog et al.,
Nucl. Acids Res. (2007) 35:1897-1907 found highly statistically significant clusters of
codons with lower than expected synonymous variation within the influenza virus
genome, located at the terminal regions of segments, where the presence of specific
packaging signals are known. Synonymous mutational analysis of these regions
confirmed the ability of their method to identify functionally significant cis-acting
elements (i.e., packaging signals) in the virus genome at the single nucleotide level.
Using these methods, then, packaging signals for the NA segment of various virus strains
and subtypes can be readily identified. Determination of packaging efficiency of
recombinant viral RNA segments can be carried out using techniques known in the art.
See, e.g., Dos et al., Virology (2005) 341:34-46.
Generally, NA packaging sequences for use in the present invention will include
at least 19 nucleotides from the 3’ UTR adjacent to the NA coding sequence, preferably
19-30 nucleotides, such as 19, 20...25...30...35 nucleotides and at least 28 nucleotides
from the 5’ UTR adjacent to the NA coding sequence, preferably 28-50 nucleotides, such
as 28, 29, 30...35...40...45...50 nucleotides. The NA packaging sequences also will
include about 145 to 250, preferably 150-200 nucleotides from at least the 3’ end of the
coding sequence and optionally from each end of the coding region for the NA segment.
Thus, for example, influenza virus packaging sequences can comprise sequences
corresponding to the 3' end of NA viral RNA including sequences corresponding to the
N-terminus of the NA coding region, e.g., at least 150 nucleotides of the 3' end of a type
A NA viral RNA such as 150, 151, 152, 153, 154,
155...160...165...170...175...180...185...190, and so on, and, optionally, packaging
sequences corresponding to the 5' end of NA viral RNA including sequences
corresponding to the C-terminus of the NA coding region, e.g., 150, 151, 152, 153, 154,
155...160...165...170...175...180...185...190, and so on.
In one particular embodiment, a construct can be provided that includes an HA
segment from one porcine influenza subtype and NA packaging sequences from another
porcine influenza subtype located 3’ and optionally 5’ to the HA segment. As described
in the examples, a construct was prepared that included an H3N2 HA flanked by H1N1
packaging sequences. This particular construct comprises an H3N2 HA sequence,
flanked by 19 nucleotides from the 3’ UTR adjacent to an H1N1 NA sequence and 183
nucleotides from the 3’ NA coding region and 28 nucleotides from the 5’ UTR adjacent
to the NA sequence and 157 nucleotides from the 5’ NA coding region. However, the
remainder of the NA coding region is absent. Typically, the packaging sequences used
are homologous to the backbone virus. Thus, if an H1N1 subtype is used as the
backbone (i.e., all H1N1 segments are present in the recombinant virus except for all or a
portion of the NA segment), NA packaging sequences from H1N1 will be retained and
the remainder of the H1N1 NA sequence conveniently replaced with an HA sequence
from a different subtype, such as an H3N2 HA sequence.
If desired, rather than a deletion, the NA coding region can be mutated such that
virus propagation is hindered. The NA region can be mutagenized in vitro by the
replacement of the appropriate nucleotides to result in the desired amino acid changes.
Such a change can include as little as one nucleotide, effecting a change in a single
amino acid, or can encompass several nucleotide changes. Mutants can be produced by
standard methods of site-directed mutagenesis. The mutations can be effected using a
mismatched primer which hybridizes to the parent nucleotide sequence (generally cDNA
corresponding to the RNA sequence), at a temperature below the melting temperature of
the mismatched duplex. The primer can be made specific by keeping primer length and
base composition within relatively narrow limits and by keeping the mutant base
centrally located. See, e.g., Innis et al, (1990) PCR Applications: Protocols for
Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468; Wu (Ed.),
Meth. In Enzymol. Vol. 217, San Diego: Academic Press (1993); Kunkel (1985) Proc.
Natl. Acad. Sci. USA, 82:488-492.
The NA mutation (e.g., deletion of all or a portion of the NA coding sequence
except the sequences harboring packaging signals) is preferably one that hinders virus
propagation.
The replication efficiency of an attenuated influenza virus may be determined, for
example, by measuring plaque size in Madin-Darby canine kidney (MDCK) cells, by
measuring virus titers over multiple growth cycles, or by isolating virus from infected
lung tissue and measuring titers. For example, an attenuated swine influenza virus of the
invention permits the attenuated virus, at a multiplicity of infection (MOI) of between
0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or a MOI of 0.0005,
0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or
6.0, to grow to titers between approximately 1 to approximately 100 fold, approximately
2 to approximately 90 fold, approximately 5 to approximately 80 fold, approximately 20
to approximately 80 fold, or approximately 40 to approximately 80 fold, approximately 1
to approximately 10 fold, approximately 1 to approximately 5 fold, approximately 1 to
approximately 4 fold, approximately 1 to approximately 3 fold, approximately 1 to
approximately 2 fold, approximately 3 to approximately 15 fold, or approximately 1, 2,
3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100
fold lower than wild-type swine influenza virus in cell culture, e.g. MDCK cells; cells of
a human (e.g., PerC6, a producer cell line derived from human embryonic retinoblasts
transformed with the E1 region of Adenovirus 5); mouse; chicken (e.g., chicken embryo
fibroblasts); rat, birds; or pig (e.g., PK(D1) cells, PK(15) cells, PK13 cells, NSK cells,
LLC-PK1 cells, LLC-PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, PK-2a/CL 13 or
SJPL cells). Replication efficiency can be determined by a hemagglutination assay of
BALF from pigs or supernatants of pig cells approximately 2 to 10 days, 3 to 7 days, 3 to
5 days, or 2, 3, 5, 6, 7, 8, 9, 10 days post-infection or when the viruses are plaqued on
MDCK cells. In one embodiment, the growth of an attenuated swine influenza virus of
the invention is compared to a particular standard or reference, e.g., wild-type swine
influenza virus A/Swine/Texas/4199-2/98. Another measure of attenuation is to grow
the virus in the absence of sialidase and measure titers as compared to a reference wild-
type strain as above.
In addition to the HA sequences, and the packaging sequences described above,
the recombinant, chimeric influenza virus will also include the remaining viral segments,
segments 1-3, 5, 7 and 8, that is, segments encoding PB2 (segment 1), PB1 (segment 2),
PA (segment 3), NP (segment 5), M1 and M2 (segment 7), NS1 and NEP (segment 8).
Nucleic acid and polypeptide sequences for these segments, as well as segments 4
(encoding HA) and 6 (encoding NA) from a number of influenza virus isolates are
known. Representative influenza sequences are presented in Figures 5-13 herein.
Additional representative sequences for the 8 influenza segments from influenza isolates
found in various species are listed in the National Center for Biotechnology Information
(NCBI) database and the Influenza Research Database found at fludb.org. See also
Ferguson et al. (2003) Nature 422: 428-433; Lin et al. (2000) Proc. Natl. Acad. Sci.
U.S.A. 97, 9654-9658; Nguyen et al. (2005) J. Virol. 79:4201-4212; Ha et al. (2002)
EMBO J. 21:865-875; and Chan et al. (2004) J. Microbiol. Immunol. Infect. 37:135-144;
for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis
of influenza virus.
Any of these sequences, as well as variants thereof can be used to produce the
recombinant, chimeric influenza viruses. Thus, the invention includes variants of the
above sequences displaying at least about 80-100% sequence identity thereto, including
any percent identity within these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity thereto.
The above-described segments can be derived from any of various swine
influenza viruses, including, without limitation, A/swine/Saskatchewan/18789/02,
A/Swine/Colorado/1/77, A/Swine/Colorado/23619/99, A/Swine/Cote d'Armor/3633/84,
A/Swine/Cote d'Armor/3633/84, A/Swine/England/195852/92,
A/Swine/Finistere/2899/82, A/Swine/Hong Kong/10/98, A/Swine/Hong Kong/9/98,
A/Swine/Hong Kong/81/78, A/Swine/Illinois/100084/01, A/Swine/Illinois/100085A/01,
A/Swine/Illinois/21587/99, A/Swine/Indiana/1726/88, A/Swine/Indiana/9K035/99,
A/Swine/Indiana/P 12439/00, A/Swine/Iowa/30, A/Swine/Iowa/15/30,
A/Swine/Iowa/533/99, A/Swine/Iowa/569/99, A/Swine/Iowa/3421/90,
A/Swine/Iowa/8548-1/98, A/Swine/Iowa/930/01, A/Swine/Iowa/17672/88,
A/Swine/Italy/1513-1/98, A/Swine/Italy/1523/98, A/Swine/Korea/CY02/02,
A/Swine/Minnesota/55551/00, A/Swine/Minnesota/593/99, A/Swine/Minnesota/9088-
2/98, A/Swine/Nebraska/1/92, A/Swine/Nebraska/209/98, A/Swine/Netherlands/12/85,
A/Swine/North Carolina/16497/99, A/Swine/North Carolina/35922/98, A/Swine/North
Carolina/93523/01, A/Swine/North Carolina/98225/01, A/Swine/Oedenrode/7C/96,
A/Swine/Ohio/891/01, A/Swine/Oklahoma/18717/99, A/Swine/Oklahoma/18089/99,
A/Swine/Ontario/01911-1/99, A/Swine/Ontario/01911-2/99, A/Swine/Ontario/41848/97,
A/Swine/Ontario/97, A/Swine/Quebec/192/81, A/Swine/Quebec/192/91,
A/Swine/Quebec/5393/91, A/Swine/Taiwan/7310/70, A/Swine/Tennessee/24/77,
A/Swine/Texas/4199-2/98, A/Swine/Wisconsin/125/97, A/Swine/Wisconsin/136/97,
A/Swine/Wisconsin/163/97, A/Swine/Wisconsin/164/97, A/Swine/Wisconsin/166/97,
A/Swine/Wisconsin/168/97, A/Swine/Wisconsin/235/97, A/Swine/Wisconsin/238/97,
A/Swine/Wisconsin/457/98, A/Swine/Wisconsin/458/98, A/Swine/Wisconsin/464/98
and A/Swine/Wisconsin/14094/99.
In one particular embodiment, an H3N2 HA is used in place of all or part of an
H1N1 NA sequence in an H1N1 backbone. Thus, the resulting recombinant virus
includes two, HAs and the remainder of the viral segments. See, Figure 1.
Each of the above described segments can be isolated from viral RNA using
known methods. For example, nucleic acids can be obtained by screening cDNA and/or
genomic libraries from cells infected with virus, or by deriving the gene from a vector
known to include the same. For example, polynucleotides of interest can be isolated
from a genomic library derived from viral RNA from an infected subject. Alternatively,
influenza virus can be isolated from infected mammals or from biological samples (e.g.,
nasal, nasopharyngeal, throat, or conjunctival secretions, blood, or anal swabs) collected
from infected subjects. Once obtained, the virus can be propagated using known
techniques, such as described in Mochalova et al., Virology (2003) 313:473-480; Lin et
al., Virology (1997) 233:402-410; Hardy et al., Virology (1995) 211:302-306; Hinshaw et
al., J. Gen. Virol. (1978) 41:115-127. Nucleic acid can also be obtained directly from
the influenza virus in question.
Thus, particular nucleotide sequences can be obtained from vectors harboring the
desired sequences or synthesized completely or in part using various oligonucleotide
synthesis techniques known in the art, such as site-directed mutagenesis and polymerase
chain reaction (PCR) techniques where appropriate. See, e.g., Sambrook, supra. One
method of obtaining nucleotide sequences encoding the desired sequences is by
annealing complementary sets of overlapping synthetic oligonucleotides produced in a
conventional, automated polynucleotide synthesizer, followed by ligation with an
appropriate DNA ligase and amplification of the ligated nucleotide sequence via PCR.
See, e.g., Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4084-4088.
Additionally, oligonucleotide directed synthesis (Jones et al. (1986) Nature 54:75-82),
oligonucleotide directed mutagenesis of pre-existing nucleotide regions (Riechmann et
al. (1988) Nature 332:323-327 and Verhoeyen et al. (1988) Science 239:1534-1536), and
enzymatic filling-in of gapped oligonucleotides using T DNA polymerase (Queen et al.
(1989) Proc. Natl. Acad. Sci. USA 86:10029-10033) can be used to produce modified
molecules.
An amplification method such as PCR can be used to amplify polynucleotides
including the various segments. In one embodiment, these segments are reverse-
transcribed into cDNA and amplified using RT-PCR. See, e.g., Hoffmann et al., Proc.
Natl. Acad. Sci. USA (2000) 97:6108-6113. The cDNA from each segment is cloned to
provide separate plasmids for use in preparing the recombinant, chimeric influenza virus.
In some embodiments, cloning vector pHW2000 (Hoffmann et al., Proc. Natl. Acad. Sci.
USA (2000) 97:6108-6113) can be used. However, the segments can be cloned into any
suitable vector. Numerous cloning vectors are known to those of skill in the art, and the
selection of an appropriate cloning vector is a matter of choice. Examples of
recombinant DNA vectors for cloning and host cells which they can transform include
the bacteriophage (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-
negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1 (gram-negative
bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus
subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5
(Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus (mammalian
cells). See, generally, DNA Cloning: Vols. I & II, supra; Sambrook et al., supra; B.
Perbal, supra.
The recombinant, chimeric, attenuated influenza virus comprising more than one
HA sequence from more than one influenza subtype can then be produced by any
method well know in the art. Preferably reverse genetics is used to produce the
recombinant viruses. Reverse genetics uses RNA polymerase complexes isolated from
influenza virus-infected cells to transcribe artificial influenza virus genome segments
containing the mutation(s). The synthesized RNA segment(s) are incorporated into virus
particles using a helper virus, and viruses containing the desired changes are then
selected. Reverse genetics methods for influenza viruses are described, for example, in
Enami et al., Proc. Natl. Acad. Sci. (1990) 87:3802 3805; Enami and Palese, J. Virol.
(1991) 65:2711-13; Luytjes, Cell (1989) 59:1107-13; Fodor et al., J. Virol. (1999)
73:9679-9682; Gao et al., J. Virol. (2008) 82:6419-6426; Quinlivan et al., J. Virol.
(2005) 79:8431-8439; and U.S. Pat. Nos. 5,578,473, 6,974,686 and 7,037,707.
Recently developed reverse-genetics systems, based entirely on cDNA, have
allowed the manipulation of the influenza viral genome. See, e.g, Palese et., Proc. Natl.
Acad. Sci. USA (1996) 93:11354; Neumann and Kawaoka, Adv. Virus Res. (1999)
53:265; Neumann et al., Proc. Natl. Acad. Sci. USA (1999) 96:9345; Fodor et al., J.
Virol. (1999) 73:9679. In one technique, modified viral RNA transcripts are transcribed
in vitro from cDNA constructs in the presence of purified NP, PB1, PB2, and PA
proteins. The resulting synthetic RNP is then transfected into cells previously infected
with an influenza helper virus. This helper virus usually has a conditional growth defect,
such as host range restriction or temperature sensitivity, which allows the subsequent
selection of transfectant viruses. For example, host-range helper viruses have been
successfully used to rescue synthetic NA and PB2 genes. See Enami, supra, and
Subbarao, J Virol (1993) 67:7223-28.
In preferred embodiments, an eight plasmid system is used to generate attenuated
influenza viruses. See, e.g., Hoffmann et al., Vaccine (2002) 20:3165-3170; Hoffmann
et al., Proc. Natl. Acad. Sci. U SA (2000) 97:6108-6113; and U.S. Patent Publication No.
20040029251. In this embodiment, the plasmids harboring the eight segments of the
desired influenza virus, such as the two HA segments, as well as segments encoding
polymerase acidic protein (PA), polymerase basic proteins 1 and 2 (PB1 and PB2), the
matrix (M) segment encoding matrix proteins 1 and 2 (M1 and M2), the nucleoprotein
(NP), and the nonstructural (NS) segment encoding nonstructural proteins 1 and 2 (NS1
and NEP), are cotransfected into an appropriate cell resulting in the recombinant,
chimeric virus described herein. See also, U.S. Patent No. 6,951,754 that describes eight
plasmid dual promoter reverse genetic systems for the production of attenuated influenza
viruses using a pol I-pol II system.
Production of live attenuated virus vaccine formulations is accomplished using
conventional methods involving propagation of the recombinant, chimeric virus in any
substrate that allows the virus to grow to titers sufficient for further use. Typically, the
viruses are propagated in cells, embryonated eggs, and/or animals followed by
purification. Generally, influenza viruses are grown in embryonated chicken eggs or
mammalian cells, such as Madin-Darby canine kidney (MDCK) cells, Madin Darby
bovine kidney (MDBK) cells, pig cells, or African green monkey kidney (Vero) cells,
using known techniques. See, e.g., Mochalova et al., Virology (2003) 313:473-480; Lin
et al., Virology (1997) 233:402-410; Hardy et al., Virology (1995) 211:302-306; Hinshaw
et al., J. Gen. Virol. (1978) 41:115-127. Representative pig cells include porcine kidney
cell lines, porcine testis cell lines and porcine lung cell lines, such as but not limited to,
PK(D1) cells, PK(15) cells, PK13 cells, SJPL cells, NSK cells, LLC-PK1 cells, LLC-
PK1A cells, ESK-4 cells, ST cells, PT-K75 cells, and PK-2a/CL 13 cells.
In another embodiment, the recombinant, chimeric swine influenza viruses are
propagated in chicken cells, e.g., chicken embryo fibroblasts derived from, e.g., 6-14
day-old embryonated eggs. In other embodiments, young or immature embryonated
eggs can be used to propagate the viruses of the invention. Immature embryonated eggs
encompass eggs which are less than ten-day-old eggs. Immature embryonated eggs may
also be eggs which artificially mimic immature eggs as a result of alterations to the
growth conditions, e.g., changes in incubation temperatures; treating with drugs; or any
other alteration which results in an egg with a retarded development. The swine
influenza viruses can be propagated in different locations of the embryonated egg, e.g.,
the allantoic cavity.
In a specific embodiment, the attenuated swine influenza viruses of the present
invention are propagated in any substrate that allows the virus to grow to titers
comparable to those determined for wild type swine influenza virus strains. Preferably,
the virions are cultured in the presence of sialidase since the NA segment in the
recombinant, chimeric virus is deficient.
It is preferred that the virus is highly purified prior to vaccine formulation.
Generally, the purification procedures will result in the extensive removal of cellular
DNA, other cellular components, and adventitious agents. Procedures that extensively
degrade or denature DNA can also be used. See, e.g., Mizrahi, ed., Viral Vaccines,
Wiley-Liss, New York (1990). Methods of purification are known in the art and may
include one or more of, for instance, gradient centrifugation, ultracentrifugation, zonal
ultracentrifugation, continuous-flow ultracentrifugation and chromatography, such as ion
exchange chromatography, size exclusion chromatography, and liquid affinity
chromatography, polyethylene glycol or ammonium sulfate precipitation.
B. Anti-Viral Compositions
The recombinant, chimeric influenza viruses, as well as recombinant, chimeric
influenza viruses that have been subsequently inactivated, can be formulated into
compositions for delivery to subjects for either inhibiting infection, or for enhancing an
immune response to influenza virus. Thus, either a live recombinant swine influenza
virus vaccine or immunogenic formulation or an inactivated recombinant swine
influenza virus vaccine or immunogenic formulation can be formulated. A live vaccine
or immunogenic formulation may be preferred because multiplication in the host leads to
a prolonged stimulus of similar kind and magnitude to that occurring in natural
infections, and therefore, confers substantial, long-lasting immunity. Production of such
live recombinant swine influenza virus vaccine formulations and immunogenic
formulations may be accomplished using conventional methods involving propagation as
described above. When formulated as a live virus vaccine, a range of about 10 to 10
3 7 4 6
can be used, preferably from about 10 to 10 , more preferably 10 pfu to about 5x10 ,
and most preferably from 10 to 10 pfu per dose should be used.
Inactivated vaccine formulations may be prepared using conventional techniques
to “kill” the attenuated viruses. Inactivated vaccines are “dead” in the sense that their
infectivity has been destroyed. Ideally, the infectivity of the virus is destroyed without
affecting its immunogenicity. In order to prepare inactivated vaccines, the attenuated
virus is grown and purified as described above. The purified virus is then inactivated
using one of several methods known in the art. Such methods include both chemical or
physical means. Chemical means for inactivating an influenza virus include treatment of
the virus with an effective amount of one or more of the following agents: detergents,
formaldehyde, formalin, ß-propiolactone, or UV light. Other methods of viral
inactivation are known in the art, such as for example binary ethylamine, acetyl
ethyleneimine, or gamma irradiation. See, e.g., U.S. Pat. Nos. 6,635,246; 5,891,705;
,106,619; and 4,693,981.
Compositions of the invention may comprise or be coadministered with a non-
influenza antigen or combination of antigens, such as with a combination influenza
vaccine. Methods of preparing such formulations are described in, e.g., Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 18 Edition,
1990. The compositions of the present invention can be prepared for mucosal delivery,
parenteral delivery, e.g., as injectables, either as liquid solutions or suspensions. Solid
forms suitable for solution in or suspension in liquid vehicles prior to injection may also
be prepared. The preparation may also be emulsified or the active ingredient
encapsulated in liposome vehicles. The active immunogenic ingredient is generally
mixed with a compatible pharmaceutical vehicle, such as, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired,
the vehicle may contain minor amounts of auxiliary substances such as wetting or
emulsifying agents and pH buffering agents.
If used to modulate an immune response, additional adjuvants which enhance the
effectiveness of the composition may also be added to the formulation. Adjuvants may
include for example, muramyl dipeptides, avridine, aluminum hydroxide,
dimethyldioctadecyl ammonium bromide (DDA), oils, oil-in-water emulsions, saponins,
cytokines, and other substances known in the art.
Carriers may also be used in order to increase the immunogenicity of the vaccine.
Suitable carriers include large, slowly metabolized macromolecules such as proteins,
including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules,
thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art;
polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like;
polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid
copolymers; and inactive virus particles.
Furthermore, influenza molecules may be formulated into compositions in either
neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts
(formed with the free amino groups of the active polypeptides) and which are formed
with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free
carboxyl groups may also be derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
Formulations will contain a “therapeutically effective amount” of the active
ingredient, that is, an amount capable of achieving the desired response in a subject to
which the composition is administered. In the treatment and prevention of influenza
infection, for example, a “therapeutically effective amount” would preferably be an
amount which prevents, reduces or ameliorates the symptoms of flu. The exact amount
necessary will vary depending on the subject being treated; the age and general condition
of the subject to be treated; the capacity of the subject's immune system to synthesize
antibodies; the degree of protection desired; the severity of the condition being treated;
the particular virus preparation selected and its mode of administration, among other
factors. An appropriate effective amount can be readily determined by one of skill in the
art. Thus, a “therapeutically effective amount” will fall in a relatively broad range that
can be determined through routine trials. The recombinant, chimeric influenza virus will
typically range from about 1% to about 95% (w/w) of the composition, or even higher or
lower if appropriate.
Additional formulations which are suitable for other modes of administration
include suppositories and, in some cases, aerosol, intranasal, oral formulations, and
sustained release formulations. For suppositories, the vehicle composition will include
traditional binders and carriers, such as, polyalkaline glycols, or triglycerides. Such
suppositories may be formed from mixtures containing the active ingredient in the range
of about 0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral vehicles
include such normally employed excipients as, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium
carbonate, and the like. These oral vaccine compositions may be taken in the form of
solutions, suspensions, tablets, pills, capsules, sustained release formulations, or
powders, and contain from about 10% to about 95% of the active ingredient, preferably
about 25% to about 70%.
Intranasal formulations will usually include vehicles that neither cause irritation
to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water,
aqueous saline or other known substances can be employed with the subject invention.
The nasal formulations may also contain preservatives such as, but not limited to,
chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance
absorption of the subject proteins by the nasal mucosa.
Controlled or sustained release formulations are made by incorporating the
protein into carriers or vehicles such as liposomes, nonresorbable impermeable polymers
such as ethylenevinyl acetate copolymers and HYTREL copolymers, swellable polymers
such as hydrogels, resorbable polymers such as collagen and certain polyacids or
polyesters such as those used to make resorbable sutures, polyphosphazenes, alginate,
microparticles, gelatin nanospheres, chitosan nanoparticles, and the like. The influenza
virus can also be delivered using implanted mini-pumps, well known in the art.
C. Administration
Compositions of the invention will generally be administered directly to a patient.
Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously,
intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue),
or mucosally, such as by intratracheal, rectal, oral (e.g. tablet, spray), vaginal, topical,
transdermal (See e.g. WO99/27961) or transcutaneous (See e.g. WO02/074244 and
WO02/064162), intranasal (See e.g. WO03/028760), ocular, aural, pulmonary or other
mucosal administration. Immunogenic compositions can also be administered topically
by direct transfer to the surface of the skin. Topical administration can be accomplished
without utilizing any devices, or by contacting naked skin with the immunogenic
composition utilizing a bandage or a bandage-like device (see, e.g., U.S. Patent No.
6,348,450).
Preferably the mode of administration is parenteral, mucosal or a combination of
mucosal and parenteral immunizations. Even more preferably, the mode of
administration is parenteral, mucosal or a combination of mucosal and parenteral
immunizations in a total of 1-2 vaccinations 1-3 weeks apart. Preferably the route of
administration includes but is not limited to oral delivery, intramuscular delivery and a
combination of oral and intramuscular delivery.
In one embodiment, the method for treating an infection by an influenza virus,
comprises mucosally administering to a subject in need thereof a first immunogenic
composition comprising the influenza viruses of the invention followed by parenterally
administering a therapeutically effective amount of a second immunogenic composition
comprising the influenza viruses of the invention.
The immunogenic composition may be used to elicit systemic and/or mucosal
immunity, preferably to elicit an enhanced systemic and/or mucosal immunity.
Preferably the immune response is characterized by the induction of a serum IgG and/or
an IgA immune response.
In any method involving coadministration, the various compositions can be
delivered in any order. Thus, in embodiments including delivery of multiple different
compositions or molecules, the influenza virus need not be delivered prior to other
immunogenic substances. For example, the priming step may include delivery of one or
more polypeptides and the boosting may comprise delivery of one or more attenutated
influenza viruses. Multiple administrations of influenza virus can be followed by
multiple administrations of other substances. Administrations can be performed in any
order. Therefore, any combination of influenza virus and other immunogenic substances
can be used to elicit an immune reaction.
D. Dosage Regime
Dosage treatment can be according to a single dose schedule or a multiple dose
schedule. Multiple doses may be used in a primary immunization schedule and/or in a
booster immunization schedule. In a multiple dose schedule, the various doses may be
given by the same or different routes, e.g. a parenteral prime and mucosal boost, a
mucosal prime and parenteral boost, etc.
Preferably the dosage regime enhances the avidity of the antibody response
leading to antibodies with a neutralizing characteristic. An in vitro neutralization assay
may be used to test for neutralizing antibodies (see for example Asanaka et al., J. of
Virol. (2005) 102:10327; Wobus et al., PLOS Biology (2004) 2; e432; and Dubekti et al.,
J. Med. Virol. (2002) 66:400).
E. Tests to Determine the Efficacy of an Immune Response
One way of assessing efficacy of therapeutic treatment involves monitoring
infection after administration of a composition of the invention. One way of assessing
efficacy of prophylactic treatment involves monitoring immune responses against the
antigens in the compositions of the invention after administration of the composition.
Another way of checking efficacy of therapeutic treatment involves monitoring
infection after administration of the compositions of the invention. One way of checking
efficacy of prophylactic treatment involves monitoring immune responses both
systemically (such as monitoring the level of IgG1 and IgG2a production) and mucosally
(such as monitoring the level of IgA production) against the antigens in the compositions
of the invention after administration of the composition. Typically, serum specific
antibody responses are determined post-immunization but pre-challenge whereas
mucosal specific antibody body responses are determined post-immunization and post-
challenge.
The immunogenic compositions of the present invention can be evaluated in in
vitro and in vivo animal models prior to host administration. The efficacy of
immunogenic compositions of the invention can be determined in vivo by challenging
animal models of infection, e.g., guinea pigs or mice or pigs, with the immunogenic
compositions. The immunogenic compositions may or may not be derived from the
same strains as the challenge strains. Preferably the immunogenic compositions are
derivable from the same strains as the challenge strains. Particularly useful mouse
models include those in which intraperitoneal immunization is followed by either
intraperitoneal challenge or intranasal challenge. In vivo efficacy mouse models include
but are not limited to a murine infection model using swine strains and a murine disease
model which is a murine model using a mouse-adapted strain, such as strains which are
particularly virulent in mice.
The immune response may be one or both of a TH1 immune response and a TH2
response. The immune response may be an improved or an enhanced or an altered
immune response. The immune response may be one or both of a systemic and a
mucosal immune response. Preferably the immune response is an enhanced systemic
and/or mucosal response.
An enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1
and/or TH2 immune response. Preferably, the enhanced immune response includes an
increase in the production of IgG1 and/or IgG2a and/or IgA. Preferably the mucosal
immune response is a TH2 immune response. Preferably, the mucosal immune response
includes an increase in the production of IgA.
Activated TH2 cells enhance antibody production and are therefore of value in
responding to extracellular infections. Activated TH2 cells may secrete one or more of
IL-4, IL-5, IL-6, and IL-10. A TH2 immune response may result in the production of
IgG1, IgE, IgA and memory B cells for future protection.
A TH2 immune response may include one or more of an increase in one or more
of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and
IL-10), or an increase in the production of IgG1, IgE, IgA and memory B cells.
Preferably, the enhanced TH2 immune response will include an increase in IgG1
production.
A TH1 immune response may include one or more of an increase in CTLs, an
increase in one or more of the cytokines associated with a TH1 immune response (such
as IL-2, IFNγ, and TNFβ), an increase in activated macrophages, an increase in NK
activity, or an increase in the production of IgG2a. Preferably, the enhanced TH1
immune response will include an increase in IgG2a production.
Immunogenic compositions of the invention, in particular, immunogenic
composition comprising one or more antigens of the present invention may be used
either alone or in combination with other antigens optionally with an immunoregulatory
agent capable of eliciting a Th1 and/or Th2 response.
The immunogenic compositions of the invention will preferably elicit both a cell
mediated immune response as well as a humoral immune response in order to effectively
address an infection. This immune response will preferably induce long lasting (e.g.,
neutralizing) antibodies and a cell mediated immunity that can quickly respond upon
exposure to one or more infectious antigens. By way of example, evidence of
neutralizing antibodies in patient blood samples is considered as a surrogate parameter
for protection since their formation is of decisive importance for virus elimination in
TBE infections (see Kaiser and Holzmann, Infection 28; 78-84).
F. Kits
The invention also provides kits comprising one or more containers of
compositions of the invention. Compositions can be in solid form, liquid form or can be
lyophilized. Suitable containers for the compositions include, for example, bottles, vials,
syringes, and test tubes. Containers can be formed from a variety of materials, including
glass or plastic. A container may have a sterile access port (for example, the container
may be an intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle).
The kit can further comprise a second container comprising a pharmaceutically-
acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose
solution. It can also contain other materials useful to the end-user, including other
pharmaceutically acceptable formulating solutions such as buffers, diluents, filters,
needles, and syringes or other delivery device. The kit may further include a third
component comprising an adjuvant.
For mucosal routes, the composition may be packaged for intranasal
administration, such as by nasal spray, nasal drops, gel or powder. See, e.g., Almeida &
Alpar, J. Drug Targeting (1996) 3:455-467; Agarwal & Mishra, Indian J. Exp. Biol.
(1999) 37:6-16 or in inhalation devices well known in the art.
The kit can also comprise a package insert containing written instructions for
methods of inducing immunity or for treating infections. The package insert can be an
unapproved draft package insert or can be a package insert approved by the Food and
Drug Administration (FDA) or other regulatory body.
The invention also provides a delivery device pre-filled with the immunogenic
compositions of the invention.
3. EXPERIMENTAL
Below are examples of specific embodiments for carrying out the present
invention. The examples are offered for illustrative purposes only, and are not intended
to limit the scope of the present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers used (e.g.,
amounts, temperatures, etc.), but some experimental error and deviation should, of
course, be allowed for.
Example 1
Generation of a Recombinant Virus including both H1 and H3 HAs
A recombinant, chimeric influenza A virus possessing, an eight segmented
genome, was produced as detailed below. The virus included seven segments of a swine
H1N1 virus with the majority of the NA segment replaced by an H3 HA coding region
sequence flanked by NA packaging sequences. The virus thus included HAs from two
different types of swine influenza, H1 HA and H3 HA. Because NA is essential for virus
propagation, the function of NA was provided by growing the virus in the presence of
sialidase (neuraminidase).
In particular, in order to generate a recombinant swine influenza virus carrying
two different HA molecules, the NA segment in an H1N1 swine influenza virus,
A/swine/Saskatchewan/18789/02, termed “SIV SK02” herein (obtained from the Prairie
Diagnostic Services, Western College of Veterinary Medicine, University of
Saskatchewan, Canada) was replaced with an H3 HA segment from the H3N2 Influenza
A virus, A/Swine/Texas/4199-2/98 (termed “SIV Tx98” herein) (Figure 1A). The H3
HA open reading frame (ORF) derived from SIV-Tx98 was flanked by NA packaging
signals that included 202nt at the 3’ end (19nt from the 3’ UTR and 183nt from the 3’
NA coding region) and 185nt at the 5’ end (28nt from the 5’ UTR and 157nt from the 5’
NA coding region) from SIV-SK02 (H1N1) strain (Figure 1B). Plasmid pHW-SIV-NA-
H3HA encoding H3 HA flanked by NA packaging signals was constructed by modifying
an original plasmid pHW-SIV/SK-NA. Briefly, the NA segment-specific packaging
signals at 3’ and 5’ ends (202nt and 185nt respectively), were amplified by polymerase
chain reaction (PCR) using pHW-SIV/SK-NA as template and the following sets of
primers: for amplifying 3’ NA packaging signal, 5'-TAATACGACTCACTATAGGG-3'
(SEQ ID NO:21) and 5’-
GTCATTTCCGGGAAGTTTTTGCACCCAAGTATTGTTTTCGTAG-3’ (SEQ ID
NO:22) were used; for amplifying 5’ NA packaging signal, 5’-
GCTGAAATCAGGATACAAAGATTGAGGCCTTGCTTCTGGGTTG-3’ (SEQ ID
NO:23) and 5’-ACAGGTGTCCGTGTCGCG-3’ (SEQ ID NO:24) were used. H3 HA
ectodomain (excluding signal peptide sequence) from SIV/Tx98 was amplified by PCR
using pHW-Tx98 HA as a template and 5’-
CTACGAAAACAATACTTGGGTGCAAAAACTTCCCGGAAATGAC-3’ (SEQ ID
NO:25) and 5’-CAACCCAGAAGCAAGGCCTCAATCTTTGTATCCTGATTTCAGC-
3’ (SEQ ID NO:26) as primers. The three pieces of PCR products were joined together
by overlapping PCR. Finally, this PCR product was digested by NaeI/NheI and replaced
the corresponding segment in pHW-SIV/SK-NA. The constructed plasmid was DNA-
sequenced to ensure that additional mutations were not introduced during the
overlapping PCRs.
This construct was inserted into cloning vector pHW2000 (Hoffmann et al., Proc.
Natl. Acad. Sci. USA (2000) 97:6108-6113) to render plasmid-606. Cloning vector
pHW2000 contains 225 bp of the human pol I promoter and 33 bp of the murine
terminator sequence separated by two BsmB1 sites. The pol I promoter and terminator
elements are flanked by a truncated immediate-early promoter of human
cytomegalovirus and by the gene encoding bovine growth hormone.
The pHW2000 vector was cotransfected with plasmid-606 and seven plasmids
which included the PB2, PB1, PA, HA, NP, M and NS segments from SIV-SK02 strain
as described in Masic et al., J. Gen. Virol. (2009) 90:375-385, in the presence of 10
mU/ml of vibrio cholera sialidase which resulted in successful rescuing of a
recombinant, chimeric virus termed “SIV/SK-606 or SIV-606” This SIV/H1H3 SIV
mutant virus was rescued using an 8-plasmid reverse genetics system described by
Hoffmann et al., Proc. Natl. Acad. Sci. USA (2000) 97:6108-6113. Briefly, 293T and
MDCK cells were co-cultured at the same density (2.5 × 10 cells/well) in a 6-well plate
and maintained in DMEM containing 10% FBS at 37ºC, 5% CO for 24 hrs. One hour
prior to transfection, medium containing FBS was replaced with fresh Opti-MEM
(Invitrogen). To rescue SIV/SK-606, cells were transfected with eight plasmid
constructs (pHW-SIV/SK-PB2, pHW-SIV/SK-PB1, pHW-SIV/SK-PA, pHW-SIV/SK-
HA, pHW-SIV/SK-NP, pHW-SIV-NA-H3HA, pHW-SIV/SK-M and pHW-SIV/SK-NS)
by Transit-LT1 transfection reagent (Mirus, Madison, Wisconsin). Six hours later, the
transfection mixture was replaced with 1ml of fresh Opti-MEM. Twenty four hours
post-transfection, one ml of Opti-MEM containing 0.4% BSA, 2µg/ml of TPCK-treated
trypsin and 10mU/ml Vibrio cholerae neuraminidase (Sigma, N6514) was added to
transfected cells. Supernatant was collected at 96 hours post transfection.
Cytopathogenic effect (CPE) was observed after the third consecutive passage on MDCK
cells and virus presence was confirmed by a hemagglutination test.
EXAMPLE 2
Characterization of SIV/SK-606
To confirm that recombinant virus SIV/SK-606 possessed both H1 and H3 HA
segments in its genome, viral RNA was isolated from purified virons. Briefly, tissue
culture grown viruses were collected by ultracentrifugation and subjected to a sucrose
gradient centrifugation (Masic et al., J. Gen. Virol. (2009) 90:375-385). For RNA
purification, purified virions were processed following manufacturer’s instruction of
Trizol (Invitrogen). Reverse transcription was performed using the Uni12 primer
(Hoffman et al., Arch. Virol. (2001) 146:2275-2289) which specifically amplifies viral
RNAs. PCR was carried out by using primers specific for H1 ( Fw 5'
TGGCCAAACCATGAGACAAC 3' (SEQ ID NO:27) and Bw 5'
GGCGTTATTCCTCAGTTGTG 3' (SEQ ID NO:28)) and H3 HAs ( Fw 5’
CGCAATCGCAGGTTTCATAG 3’ (SEQ ID NO:29) and Bw 5’
CAACCCAGAAGCAAGGCCTCAATCTTTGTATCCTGATTTCAGC 3’ (SEQ ID
NO:30)).
While PCR products representing the H1 HA segment were detected only in the
SK02 and the SIV-606 genomes, PCR bands representing the H3 HA segment were
observed in Tx98 and SIV-606 viral RNA extraction. These data demonstrated that the
genome of SIV-606 included both H1 and H3 HA segments.
To examine whether both HAs were expressed, viral infected cell lysates were
subjected to Western blotting analysis using antibodies specific for H1HA, H3HA and
M1. Briefly, MDCK cells were infected with wild-type SIV/SK02, wild-type SIV/Tx98,
or SIV-606 at an MOI of 0.01. At 48 hours post-infection, cell lysates were prepared and
were subjected to Western blotting analysis using antibodies specific for H1 HA (Anti-
HA (A/California/06/2009( (H1N1) monoclonal antibody, eEnzyme (Maryland, USA),
H3 HA (Anti-multi-Hemagglutinins (H3N2) Antibody, rabbit IgG, eEnzyme (Maryland,
USA) and M1 (Shin et al., J Gen Virol (2007) 88:942-950).
M1 protein was detected in all virus infected cells, however, H1 HA was seen
only in SK02 and SIV-606 infected cells and H3 HA was seen in Tx98 and SIV-606
samples. Together, these data demonstrate that the H3 segment was incorporated into
the genome of SIV-606 and both HAs were expressed.
To observe the morphology of the recombinant virus, negative staining
transmission electron microscopy was performed. The majority of virions exhibited
spherical enveloped particles of approximately 100 nm in diameter, which resembled the
morphology of the wild type virus.
The replication potential of the SIV-606 was investigated in MDCK cells. In the
presence of sialidase, SIV-606 formed plaques similar in size as wild-type virus. In
contrast, SIV-606 did not grow in the absence of sialidase, indicating that replication of
the recombinant virus was dependent on sialidase. The growth potential and kinetics of
SIV-606 were also determined. As shown in Figure 2, SIV-606 reached a plateau at 24
h.p.i. as did the wild-type virus. SIV-606 grew to a titer of 7x 10 PFU/ml, which was
approximately 1 log lower than wild-type virus. These results indicated that although
SIV-606 had a slightly lower titer, it grew to relatively high titer in cell culture, which
enables propagation of the virus.
EXAMPLE 3
Pathogenicity of SIV-606 in Pigs
The pathogenicity of SIV-606 was evaluated in pigs. Thirty-five 4-week old
SIV-negative pigs were split randomly into seven groups of five pigs. These were
infected intratracheally with 4 ml MEM containing 1x10 or 1x10 PFU/ml SK02 WT,
SIV-606 or Tx98. The animals in the control group were mock infected with medium
only (Table 1). Rectal temperature was monitored daily. On day 5 post infection pigs
were euthanized and necropsies were performed. As shown in Figures 3A-3C, pigs
infected with wild-type viruses had an increased temperature on day 1 post infection, and
the temperature decreased gradually on the following days. However, pigs infected by
SIV-606 did not show elevated temperatures compared to the control group. At
necropsy, the macroscopic lung lesions were documented. The mock, SIV-606 high
dose- and low dose-infected pigs did not show any typical macroscopic lung lesions. In
contrast, gross lesions characterized as purple- to plum-colored consolidated areas were
observed in cardiac lobes of pigs infected by SK02 and Tx98 with high and low doses.
In agreement with these results, SK02 wild type virus could be recovered from lung
tissue of all animals infected with low and high doses of SK02 with median titers of
2.4x10 PFU/ml and 2.6x10 PFU/ml respectively. Similarly, wild type virus could be
isolated from lung tissue of all pigs infected with Tx98 virus with median titers of 1x10
PFU/ml and 3.4x10 PFU/ml in low and high dose groups. However, SIV-606 virus was
only detected from one pig in the low dose group and 3 pigs from the high dose group
with a very low titer (median titers were 0 and 20 PFU/ml respectively). These results
demonstrated that the SIV-606 virus is highly attenuated in pigs and thus can be used as
a live, attenuated vaccine for swine influenza.
Table 1. Assignment of pigs, dose and route of virus infection
Group N=5 Inoculum Concentration Dose Volume Route
1 MEM 4ml Intratracheal
2 SK02-WT 10 PFU/ml 4ml Intratracheal
3 SK02-WT 10 PFU/ml 4ml Intratracheal
4 SIV-606 10 PFU/ml 4ml Intratracheal
SIV-606 10 PFU/ml 4ml Intratracheal
6 Tx98 10 PFU/ml 4ml Intratracheal
7 Tx98 10 PFU/ml 4ml Intratracheal
EXAMPLE 4
Protective Effect of SIV-606 in Pigs
To determine whether SIV-606 was immunogenic and could provide protection
from SIV infection, the following vaccination and viral challenge studies were
performed in pigs. Twenty three H1N1 and H3N2 sero-negative pigs were randomly
divided into five groups (n=5, except n=3 in group 5) (Table 2). Two groups of pigs
were given MEM and two groups of pigs were vaccinated with 4 × 10 PFU of SIV-606
virus intratracheally. Pigs received a second vaccination on day 21. Ten days after the
second vaccination (on day 31), pigs were challenged intratracheally with either
SIV/SK02 or SIV/Tx98 and were euthanized on day 5 post infection. Sera were
collected prior to the first vaccination, 21 days after the first vaccination and 10 days
after the second vaccination (before viral challenge). Antigen specific serum IgG and
nasal IgA were measured on day 0, 21 and 31.
After the first vaccination, SIV/SK02 specific IgG in serum increased
significantly and the second dose of SIV-606 boosted IgG response measured on day 31
(Figure 14A). Serum IgG against SIV/Tx98 or a heterologous H1N1 Halifax210 strain,
which was isolated during a 2009 pandemic, increased slightly after one vaccination and
increased significantly after the second vaccination (Figures 14B and 14C).
To assess the presence of IgA antibodies specific for H1N1 and H3N2 influenza
viruses at mucosal surfaces in the upper and lower respiratory tract, nasal swabs and
bronchoalveolar lavage fluid (BALF) samples from pigs in all groups were collected and
tested by ELISA. The first vaccination of SIV-606 induced moderate IgA levels in nasal
secretion and the second vaccination boosted IgA induction specific to SIV/SK02,
SIV/Tx98 and Halifax210 (Figures 15A, 15B and 15C). Similarly, IgA titers remained
low in BALF after the first vaccination and were significantly higher after the second
vaccination (Figures 16A, 16B and 16C).
After the viral challenge on day 31, rectal temperature was measured daily for 5
days until necropsy. On day 1 post infection, the pigs vaccinated with MEM and
challenged with SIV/SK02 had an onset of fever with a mean rectal temperature of 40.9
°C. In contrast, pigs vaccinated with SIV-606 and challenged with SIV/SK02 had a
normal temperature ranging between 39.1 °C to 39.6 °C during these five days (Figure
17A). Similarly, the temperature of pigs vaccinated with MEM and challenged with
Tx98 rose to 40.1°C on day 1 post infection then decreased to 39.6 °C the following day
and went back to 39.3 °C on day 5 post infection. Fever was not seen in pigs vaccinated
with SIV-606 and challenged with Tx98 (Figure 17B). The temperature in this group
fluctuated between 39.2 °C and 39.7 °C during the 5 days post infection.
At necropsy, SIV-induced gross lung lesions were examined and scored by the
percentage of surface that lesions took up compared to the total lung area (Figure 18A).
All pigs in the unvaccinated groups and challenged with SIV/SK02 or SIV/Tx98
manifested SIV typical gross lesions seen as clear demarcation of dark purple,
consolidated areas mostly found in the apical and cardiac lobes. The mean score for
these two groups were 8.6 and 14.6, respectively. In contrast, the lungs of pigs
vaccinated with SIV-606 and challenged with either SIV/SK02 or SIV/Tx98 had no
gross lung lesions.
To measure the viral load in the lungs (Figure 18B), tissues from the right apical,
cardiac and diaphragmatic lobes were collected at necropsy. Virus was detected in the
lung tissues from all pigs in the unvaccinated and SIV/SK02 challenged group (mean
viral titer was 1.90 × 10 PFU per gram). In the unvaccinated and SIV/Tx98 challenged
group, virus was only isolated from one pig. No virus was detected in the lung tissues of
pigs vaccinated with SIV-606 and challenged with SIV.
Histophathological lesions were examined using lung tissue samples taken from
the right apical, cardiac, and diaphragmatic lobes at necropsy. As shown in Figure 19B
and 19D, pathological lesions were observed in the lung tissues of unvaccinated and
virus challenged groups. The histophathological lesions included the loss of bronchial
epithelium due to the necrosis of bronchiolar epithelium, hypertrophy and hyperplasia of
bronchiolar epithelium to compensate for the necrosis of bronchiolar epithelium,
neutrophil infiltration through the mucosa and into the lumen of bronchioles,
peribronchiolar and perivascular lymphocyte infiltration, interstitial thickening, and
proliferation of the bronchiolar associated lymphoid tissues. In contrast, no
histopathological changes were observed in the lung tissues of SIV-606 vaccinated and
challenged groups (Figures 19C and 19E). Both SIV-606 vaccinated groups maintained
healthy bronchiolar epithelium and alveolar structures with mild interstitial thickening,
similar to the tissue shown in the unvaccinated and unchallenged group (Figure 19A).
Table 2. Assignment of pigs for virus challenge
Vaccination
Challenge
Group
(day 31)
1 (day 0) 2 (day 21)
1 (n=5) MEM MEM SIV/SK02
2 (n=5) MEM MEM SIV/Tx98
3 (n=5) SIV-606 SIV-606 SIV/SK02
4 (n=5) SIV-606 SIV-606 SIV/Tx98
(n=3) - - -
Thus, recombinant, chimeric influenza viruses are disclosed, as well as
compositions and methods for treating and preventing influenza. Although preferred
embodiments of the subject invention have been described in some detail, it is
understood that obvious variations can be made without departing from the spirit and the
scope of the invention as defined by the appended claims.
701802/91
Claims (3)
1. A recombinant, chimeric influenza A virus comprising eight segments, wherein said virus comprises more than one hemagglutinin (“HA”) segment (“segment 4”) from more than one influenza subtype, wherein said virus comprises segments 1-5, 7 and 8 from a porcine H1N1 influenza subtype and a second segment 4 from a porcine H3N2 influenza subtype, wherein the H3N2 segment 4 comprises neuraminidase (“NA”) packaging sequences from said H1N1 influenza subtype located 3’ to said second segment 4, and further wherein all or a portion of the NA segment (“segment 6”) of the H1N1 influenza subtype is missing to render an attenuated virus.
2. The recombinant, chimeric influenza virus of claim 1, comprising 3’ NA packaging sequences from the 3’ NA UTR and the 3’ NA coding sequence and, 5’ NA packaging sequences from the 5’ NA UTR and the 5’ NA coding sequence, located 5’ to said second segment 4.
3. The recombinant, chimeric influenza virus of claim 1 or 2, wherein the H1N1 subtype is A/swine/Saskatchewan/
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161514156P | 2011-08-02 | 2011-08-02 | |
US61/514,156 | 2011-08-02 | ||
PCT/CA2012/000717 WO2013016805A1 (en) | 2011-08-02 | 2012-07-31 | Recombinant swine influenza virus and uses thereof |
Publications (2)
Publication Number | Publication Date |
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NZ620275A NZ620275A (en) | 2016-03-31 |
NZ620275B2 true NZ620275B2 (en) | 2016-07-01 |
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